Autotrophic microorganisms, the pivotal carbon fixers, exhibit a broad distribution across diverse environments, playing critical roles in the process of carbon sequestration. However, insights into their distribution characteristics in aquifers, particularly in those petroleum-hydrocarbon-contaminated (PHC) aquifers that were known for rich in heterotrophs, have been limited. In the study, groundwater samples were collected from red clay aquifers in the storage tank leakage area of a PHC site, a prevalent aquifer type in southern China and other regions. Metagenomics combined with hydrochemical and inorganic carbon isotope analyses were employed to elucidate the presence of microbial carbon fixation and its driving forces. Results showed that there were hundreds of autotrophic microorganisms participating in distinct carbon fixation processes in the red clay PHC aquifers. Reductive tricarboxylic acid (rTCA) and dicarboxylate/4-hydroxybutyrate (DC/4HB), as well as 3-hydroxypropionate (3HP or/and 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB)) were the predominant carbon fixation pathways. The abundances of carbon fixation genes and autotrophic microorganisms were significantly and positively correlated with hydrocarbon concentrations and δ 13 C of dissolved inorganic carbon (δ 13 C-DIC) values. This finding indicated that the petroleum hydrocarbon significantly promoted the proliferation of carbon fixation microorganisms, leading to a substantial uptake of inorganic carbon. Therefore, we deduce that this process holds considerable potential for carbon sequestration in PHC-contaminated aquifers.