Knowledge of the influence of grassland-to-cropland conversion on subsoil carbon (C) and nitrogen (N) stocks and the effect of soil properties on subsoil C and N stocks after grassland cultivation remain poorly understood. Furthermore, soil C and N changes retain a biological coupling under global changes; however, whether the coupling between soil C and N stocks can be disrupted by long-term grassland cultivation remains largely unknown. Here, we explored changes in soil C and N stocks at three soil depths (0-10, 10-30, and 30-50 cm) across a grassland cultivation chronosequence of 50 years in a semiarid agro-pastoral ecotone in northern China. Our results showed that soil C and N stocks in the 0-10 cm soil layer exhibited a valley-shaped pattern with increasing cultivation duration, reaching the lowest value at 20 years of grassland cultivation. For the 10-30 cm and 30-50 cm soil layers, soil C and N stocks within 10 years of grassland cultivation were higher than those in native grassland. Soil C and N stocks bottomed out at 20 years of grassland cultivation and then increased with the duration of grassland cultivation. Cultivation duration had no direct effect on soil C and N stocks across soil depths but exerted an indirect influence by affecting soil properties. Soil properties mainly drove variations in C and N stocks in soil, which strengthened with soil depth. Soil C and N stocks in the 0-10 cm soil layer showed a positive correlation with soil pH and soil clay and silt contents and were negatively related to soil sand content. Soil C and N stocks in the 10-30 and 30-50 cm soil layers were only positively correlated with soil clay content. These indicate that soil clay plays a vital role in regulating variations in C and N stocks in topsoil and subsoil with the duration of grassland cultivation. Moreover, soil C stocks in the three soil layers exhibited a significant correlation with soil N stocks. This reveals that the coupled correlation between soil C and N stocks still remains under long-term grassland cultivation.