Adopting an optimal cropping regime is crucial for sustainable soil use. However, how different cropping regimes impact phosphorus (P) availability and the underlying mechanism remain unclear. Here, a 10-year field experiment was performed to examine the influence of different cropping regimes, including maize–soybean rotation (MSR), continuous maize cropping (CMC), and farmland fallow (FALL), under unfertilized and fertilized conditions in Northeast China. The P forms were analyzed using chemical fractionation and solution phosphorus-31 nuclear magnetic resonance. Compared to FALL, total P and different forms of P contents were significantly lower under MSR and CMC systems. Moreover, the contents of total P and different forms of P were higher under MSR than those under CMC. Correlation analysis showed that there were significant and positive correlations between total P and different forms of P contents. Redundancy analysis revealed soil organic carbon (SOC) as the most significant factor influencing total P and different forms of P. Structural equation modeling demonstrated the direct positive impacts of SOC, total nitrogen, total phosphorus, and Olsen phosphorus on phosphatase activity, which exhibited direct positive influence on P availability. In summary, maize–soybean rotation is an effective cropping regime for promoting P accumulation and availability in this region.