The soybean seed yield response to phosphorus (P) fertilizer has significantly increased during soybean breeding but the underlying mechanisms remain elusive. In this study, we used two soybean cultivars with high (Andou 8 (AD8)) and low (Qiandou 5 (QD5)) seed yield response to P fertilizer to compare their yield and yield components, biomass accumulation and partitioning, P content and partitioning and P-use efficiency in Shiqian (SQ) and Dafang (DF) during the growing season in 2020 under 0 (P0) and 35 (P35) kg P ha−1 supply. The results show that AD 8 had a greater seed yield, pod biomass, filled-pod number, seed number, pod P content and P partitioning to pods, and P-use efficiency based on seed yield (PUESY) but lower P-use efficiency based on biomass (PUEB) than QD5 at two experimental sites. The seed number was positively correlated with total biomass and P content, especially with greater pod biomass accumulation and its partition to pods which showed a positive correlation with seed number. Seed number was negatively correlated with stem dry weight and its partitioning to stem, and stem P partition. We conclude (1) different yield response to P fertilizer are explained by different biomass and P content and their partitioning to pod; (2) high biomass and P-conversion efficiency increased the seed number, which accounted for a high yield response to P fertilizer. Our results highlight that soybean breeding increased biomass and nutrient content and their conversion efficiency to achieve high seed yield with P-fertilizer supply.