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Organism using inorganic substrate to obtain reducing equivalents for use in biosynthesis
Lithotrophs are a diverse group of organisms using an inorganic substrate (usually of mineral origin) to obtain reducing equivalents for use in biosynthesis (e. g., carbon dioxide fixation) or energy conservation (i. e., ATP production) via aerobic or anaerobic respiration. While lithotrophs in the broader sense include photolithotrophs like plants, chemolithotrophs are exclusively microorganisms; no known macrofauna possesses the ability to use inorganic compounds as electron sources. Macrofauna and lithotrophs can form symbiotic relationships, in which case the lithotrophs are called "prokaryotic symbionts". An example of this is chemolithotrophic bacteria in giant tube worms or plastids, which are organelles within plant cells that may have evolved from photolithotrophic cyanobacteria like organisms. Chemolithotrophs belong to the domains Bacteria and Archaea. The term "lithotroph" was created from the Greek terms 'lithos' (rock) and 'troph' (consumer), meaning "eaters of rock". Many but not all lithoautotrophs are extremophiles. The last universal common ancestor of life is thought to be a chemolithotroph (due to its presence in the prokaryotes). Different from a lithotroph is an organotroph, an organism which obtains its reducing agents from the catabolism of organic compounds.

The term was suggested in 1946 by Lwoff and collaborators.

Lithotrophs consume reduced inorganic compounds (electron donors).

A chemolithotroph is able to use inorganic reduced compounds in its energy producing reactions. This group of organisms includes sulfur oxidizers, nitrifying bacteria, iron oxidizers, and hydrogen oxidizers. The term "chemolithotrophy" refers to a cell's acquisition of energy from the oxidation of inorganic compounds, also known as electron donors. This form of metabolism is believed to occur only in prokaryotes and was first characterized by Ukrainian microbiologist Sergei Winogradsky.

The survival of these bacteria is dependent on the physiochemical conditions of their environment. Although they are sensitive to certain factors such as quality of inorganic substrate, they are able to thrive under some of the most inhospitable conditions in the world, such as temperatures above 110 degrees Celsius and below 2 pH. The most important requirement for chemolithotropic life is an abundant source of inorganic compounds, which provide a suitable electron donor in order to fix CO2 and produce the energy the microorganism needs to survive. Since chemosynthesis can take place in the absence of sunlight, these organisms are found mostly around hydrothermal vents and other locations rich in inorganic substrate. The energy obtained from inorganic oxidation varies depending on the substrate and the reaction. For example, the oxidation of hydrogen sulfide to elemental sulfur by ½O2 produces far less energy (50 kcal/mol or 210 kJ/mol) than the oxidation of elemental sulfur to sulfate (150 kcal/mol or 627 kJ/mol) by 3/2 O2,. The majority of lithotrophs fix carbon dioxide through the Calvin cycle, an energetically expensive process.

Biogeochemical cycling of elements is an essential component of lithotrophs within microbial environments. For example, in the carbon cycle, there are certain bacteria classified as photolithoautotrophs that generate organic carbon from atmospheric carbon dioxide. Certain chemolithoautotrophic bacteria can also produce organic carbon, some even in the absence of light. Lithotrophs play an important role in the biological aspect of the iron cycle. These organisms can use iron as either an electron donor, Fe(II) → Fe(III), or as an electron acceptor, Fe (III) → Fe(II). Another example is the cycling of nitrogen. Many lithotrophic bacteria play a role in reducing inorganic nitrogen (nitrogen gas) to organic nitrogen (ammonium) in a process called nitrogen fixation. Furthermore, organic components (biosignatures) that are often associated with biominerals are believed to play crucial roles in both pre biotic and biotic reactions. On January 24, 2014, NASA reported that current studies by the Curiosity and Opportunity rovers on Mars will now be searching for evidence of ancient life, including a biosphere based on autotrophic, chemotrophic and/or chemolithoautotrophic microorganisms, as well as ancient water, including fluvio lacustrine environments (plains related to ancient rivers or lakes) that may have been habitable. The search for evidence of habitability, taphonomy (related to fossils), and organic carbon on the planet Mars is now a primary NASA objective.