The increase in atmospheric carbon dioxide (CO₂) concentration and global climate change are the most important environmental challenges we faced. Autotrophic bacteria, with the capability of CO₂ assimilation, are widespread in diverse environments, thus studies on molecular mechanisms of autotrophic bacteria CO₂ fixation are of great scientific importance. Previous researches were mainly based on genomic DNA, revealing the diversity and composition of CO₂ fixation microorganism in diverse ecological systems. However, the functional implication of these autotrophies is still unknown. Transcriptomics technique and stable isotope probe technique (SIP) are successfully employed in recent studies concerning the ecological function of CO₂ fixation microorganism, providing unequivocal evidence for CO₂ assimilation by autotrophic microorganisms in complex aquatic environments and identifying new autotrophic bacteria capable of CO₂ assimilation. Therefore, the microbial mechanisms of CO₂ fixation by autotrophic mciroorganism based on SIP are summarized and the methodological considerations of RNA-SIP for molecular ecology of carbon fixation in agricultural ecosystems are presented. The translocation and stability of assimilated carbon were also discussed. This paper will offer the theoretical basis of terrestrial ecosystem carbon cycle and response to climate change.