In order to investigate the effect of restoring rice-upland rotation on soil phosphorus (P) availability in rice-crayfish co-culture paddies, three field types: rice-upland rotation restored from rice-crayfish co-culture (IRCC-PR), continuous rice-crayfish co-culture (IRCC), and traditional rice-upland rotation (PR) were selected in Yanwo Town, Honghu City, Hubei Province. Soil samples were collected from 0 to 50 cm depth, and soil available P contents and alkaline phosphatase activities (APA) were measured. The modified Hedley P fractionation method was used to determine various soil P fractions. Results showed that moderate P (Moderate-P) had the highest proportion (57.2%-72.7%), followed by stable P (Residual-P, 25.5%-40.0%), with labile P (Labile-P) had the lowest proportion (1.8%-5.4%). Compared to IRCC and PR, IRCC-PR showed increased proportions of Labile-P and Residual-P, while the proportion of Moderate-P decreased. Similar to PR, IRCC-PR exhibited an obvious surface accumulation pattern for available P and APA, their concentrations in surface soils (0-5 cm) were 5.1 and 3.0 times higher than those in subsurface soils (30-50 cm), respectively. In contrast, these parameters slightly increased with depth in IRCC. In conclusion, the results revealed that the restoration of rice-upland rotation altered the vertical distribution of soil available P, emphasizing the need to shift the risk control strategy for P loss in paddy fields from managing leaching losses to controlling P loss with soil erosion and surface runoff.