Global agricultural intensification leads to a decline in soil quality; however, the extent to which long-term rice cultivation adversely impacts soil, based on chemical and microbial perspectives, remains unclear. The present study was conducted on a seed multiplication farm in Wuchang, Heilongjiang Province, China, to quantify changes in the nutrient properties and microbial profiles of meadow soil in cultivated (rhizosphere and bulk soil) and uncultivated paddy plots from spring to winter. A non-parametric method was used to compare carbon metabolism characteristics among the three groups of soil samples. Principal component analysis was used to distinguish soil chemical properties and carbon source utilization profiles among the soil samples across different seasons. Under rice cultivation, pH, organic matter, total nitrogen, and alkali-hydrolyzed nitrogen concentrations were generally higher in rhizosphere soils than in bulk or uncultivated soils. However, microbial biomass in cultivated soils was consistently lower than in uncultivated soils. There was a discernible difference in carbon substrate preference between summer and other seasons in the three sample groups. In conclusion, agricultural activities in rice cultivation could reshape soil microbial communities in the long term. Notably, specific cultivation activity may induce distinct soil microbial responses, which are more sensitive than chemical responses.