Soil microorganisms play important roles in nutrient cycling and ecosystem functioning. Knowledge on the responses of soil microbial metabolic characteristics to agricultural practices is critical to better understand soil microbial processes in agroecosystems. The study aimed to explore the responses of soil microbial metabolic activities and their carbon utilization under different tillage and crop succession systems in a 12-year field experiment in a clay loam soil of Jilin Province, China. This experiment was set up in a split-plot design with four replicates including no-tillage (NT) and conventional tillage (CT) as main plots and continuous corn (CC) and corn-soybean succession (CS) treatments as subplots. Soil samples were collected from 0 to 5 cm and 5–15 cm depths. The results showed that compared with CT, NT increased soil organic carbon, soil microbial biomass carbon, basal respiration and microbial coefficient at 0–5 cm depth. Greater basal respiration was observed under CC than under CS at both depths. Compared with NTCC, NTCS decreased microbial functional diversity at 0–5 cm depth and increased microbial metabolic quotient at 5–15 cm depth. Redundancy analysis indicated that crop succession management induced the changes in soil microbial carbon utilization. Compared with other soil environmental factors, NO3−-N and soil moisture significantly affected soil microbial metabolic activities. No significant correlation was found between microbial carbohydrate utilization and soil organic carbon in this study. Structural equation modeling analysis suggested that corn-soybean succession practice could stimulate the consumption of aromatic acids, which indirectly affected the accumulation of soil organic carbon.