Soybean and maize rotation has been recommended as a promising strategy to maximize crop yield. However, the impacts of soybean-maize rotation, and particularly the frequency of soybean inclusion, on soil quality and carbon (C) stock need to explored. We conducted an 8-year field experiment of randomized design in Northeast China with four cropping systems as continuous soybean (S), continuous maize (M), soybean-maize rotation (SM), and soybean-maize-maize rotation (SMM). The results showed that the soil quality index, soil ecosystem multifunctionality (EMF), and C stock under crop rotation (SM and SMM) were 5.4–23.5%, 13.1–22.6%, and 9.3–29.4% higher than those under continuous cropping (M and S), respectively. Additionally, the increased frequency of soybean in the rotation increased soil EMF by 14.8% due to higher soil enzyme activities and available N. Notably, the soybean-maize rotation alleviated microbial nitrogen (N) limitation compared to continuous cropping, due to stimulated C, N, and P acquisition by enzyme activities. Furthermore, the soil quality index correlated positively with C stock in the topsoil. The accumulation rates of soil organic C and total N were higher by 0.39 and 0.14 g kg􀀀 1 year􀀀 1 in SMM than in SM, respectively. Therefore, scientifically based soybean frequency is an effective approach to enhance soil organic C in soybean and maize rotation. In conclusion, soybean-maize rotation improves soil quality compared to monoculture, and a reduced frequency of soybean within the rotation (SMM) is beneficial for C and N storage.