Soil autotrophic bacteria are important for sequestrating atmospheric CO 2 and thus contribute to the global carbon cycle. However, information is limited on how chemical fertilization can alter the composition of soil autotrophic bacteria. In this study, the cbbL gene (a key gene in the Calvin-Benson-Bassham cycle that encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, RubisCO)was used to study the abundance and composition of autotrophic bacteria in an entisol with 26 years of continuous fertilization. In this process, five chemical fertilization regimes were examined: without fertilization control (CT), nitrogen and phosphorus fertilization (NP), N and potassium fertilization (NK), PK fertilization (PK), and NPK fertilization (NPK). The results showed that after the 26-year (1991–2017)period, the CT with a neutral pH (7.55)had a higher RubisCO activity than did the chemical fertilizer treatments, but the cbbL abundance was the lowest under CT. Soil RubisCO activity positively correlated with soil pH (R 2 = 0.67, P < 0.001); soil cbbL abundance significantly positively correlated with soil available N (R 2 = 0.54, P < 0.01)and significantly negatively correlated with pH (R 2 = 0.30, P < 0.05). Meanwhile, the soil RubisCO activity significantly negatively correlated with cbbL abundance (R 2 = 0.67, P < 0.001). In addition, the alpha-diversity of cbbL was lowest under CT, indicating a positive effect of fertilization on the soil cbbL diversity. In conclusion, a 26-year long-term chemical fertilization changed the community structure of soil cbbL-carrying bacteria, and soil pH and available N played crucial roles in controlling the ecological properties of soil cbbL-carrying bacteria.