Background and aims <p>Soil microbial community structure and diversity are critical for maintaining the health of soil ecosystems. However, how soil microbes regulate the soil carbon cycle under long-term greenhouse cultivation remains unclear.</p> Methods <p>We used macro-genomics to analyze changes in microbial community structure and diversity as well as carbon cycle-related functional genes in a long-term greenhouse-grown soil.</p> Results <p>Long-term greenhouse cultivation significantly altered soil microbial community structure, manifested by decreased bacterial diversity and increased fungal diversity. The rTCA cycle served as the dominant carbon fixation pathway, with microbial carbon fixation capacity markedly reduced during the initial greenhouse phase but gradually restored as cultivation duration extended. Prolonged greenhouse practices elevated the abundance of degradation genes for labile carbon (i.e. starch) while suppressing those for recalcitrant carbon (i.e. cellulose and lignin). Soil pH is the primary driver of changes in microbial community structure and shifts in carbon cycling functional genes.</p> Conclusions <p>Long-term greenhouse cultivation reshaped the microbial community structure by altering soil properties, thereby driving adaptive shifts in microbial carbon cycling functions. The findings provide new insights into the microbial mechanisms underlying soil carbon cycling in long-term greenhouse vegetable cultivation.</p>