In many temperate soybean [Glycine max (L.) Merr.] production systems sowing date expands for several months. Early sowing dates commonly show maximum yields, but their consequences for crop N uptake and biological nitrogen fixation (BNF) are unknown. Our objective was to evaluate the role of sowing date, a major management variable defining soybean yield, on BNF. We hypothesized BNF is maximum at intermediate sowings because BNF is limited by low soil temperatures at early sowing dates and by high soil nitrate concentrations at later sowings. Four genotypes (maturity groups [MGs] IV and V) were tested over a 16‐wk sowing date period, and a N fertilizer treatment at sowing was added in all sowing date by genotype combinations (0 and 100 kg N ha–1). The N fertilization treatment helped to test sowing date effects independently of soil N availability. Seed yield and total N uptake were affected by sowing date and genotype, but not by the N treatment. Both seed yield and total N uptake followed similar quadratic responses, but MG V genotypes maximized yield and N uptake at the earliest sowing while MG IV cultivars maximized both traits at intermediate sowings. However, delaying sowing date always decreased the proportion of total aboveground N that came from BNF (average decline 0.22% d–1). Nitrogen fertilization reduced BNF by 15% across sowing dates and genotypes, with no yield effect. The linear decline in BNF with delayed sowing dates, instead of an optimum response, supports the concept that soil nitrates are the main driver of soybean BNF in this production system.