Autotrophic microorganisms, with their remarkable CO₂ sequestration capacity and exceptional environmental adaptability, play a pivotal role in driving natural carbon cycling and mitigating global climate change. However, the carbon fixation mechanisms and contributions of microorganisms have remained unclear across different ecosystems. Building on recent research findings, this paper reviewed the carbon sequestration pathways and molecular mechanisms of autotrophic microorganisms, and provided an in-depth analysis of key microbial carbon fixation pathways, community compositions, sequestration rates, and influencing factors in aquatic ecosystems (e.g., oceans, glaciers) and terrestrial ecosystems (e.g., farmlands, deserts, karst regions, forests, and grasslands). It found that there are seven CO₂fixation pathways for microorganisms in nature, among which the Calvin (CBB) cycle is particularly widely distributed in various habitats, while non-CBB cycles (such as rTCA, WL cycle, etc.) have long been overlooked for microbial carbon sequestration. There were significant differences in the carbon sequestration mechanisms and rates of microorganisms in different ecosystems, with climate factors, soil properties, agricultural activities, and other key factors regulating carbon sequestration rates. However, a quantitative evaluation of the extent to which different factors regulate microbial carbon sequestration potential was lacking. According to preliminary estimates, the carbon sequestration flux of terrestrial microorganisms may be higher than that of the ocean. However, most research on microbial autotrophic carbon sequestration was still in the qualitative or quantitative analysis stage of characteristic enzymes or genes, and there was still a lack of systematic evaluation of microbial carbon sequestration rate and flux data. Therefore, there is still significant uncertainty in the estimation of global autotrophic microbial carbon sinks. Grounded in the theoretical framework of “energy-environment” interactions, this article revealed ecological niche differentiation characteristics and potential contributions of different autotrophic carbon-fixing microbial pathways, and it also provided a novel perspective for accurately assessing global microbial carbon sink potential and offered significant guidance for developing microbial-based “carbon neutrality” technologies.