Organic fertilizer substitution is an effective measure for increasing both the quantity and quality of wheat grain while reducing chemical fertilizer input. However, the effects of reducing nitrogen (N) fertilizer combined with organic fertilizer substitution on grain yield, grain protein content and protein yield, plant N accumulation and translocation, N use efficiency, soil fertility, N apparent surplus and nitrate-N residue in rain-fed drought-prone areas remains limited. In this study, field experiments were conducted over four consecutive seasons (2019-2023) at two sites with four treatments: zero N application (ZN), farmer N application (FN), reduced 20% N of FN (RN), and organic fertilizer substituting 20% N of RN (OSN). The results showed that compared with the ZN treatment, the FN, RN and OSN treatments increased grain yield and its components, grain protein content and protein yield, aboveground N accumulation at the anthesis and maturity stages, pre-anthesis N translocation, post-anthesis N accumulation, N use efficiency, soil fertility. Compared with RN and FN, OSN increased grain yield by 17.12% and 15.03%, grain protein yield by 3.31% and 17.15%, grain N accumulation by 17.78% and 15.58%, and N harvest index by 2.63% and 4.45% averaged across years and sites, respectively. Moreover, OSN increased the contents of organic matter, total N, available P and available K in both 0-20 and 20-40 cm soil layers, decreased N apparent surplus and nitrate-N residue in 0-100 cm, and pH in both 0-20 and 20-40 cm soil layer. Fundamentally, this study suggests that integrating a 20% reduction N from conventional farmer practices with the utilization of organic fertilizer to replace 20% of the chemical N fertilizer (OSN) represents an effective strategy. This approach shows promise in enhancing wheat grain yield, grain protein yield, and N use efficiency. Additionally, it supports the improvement of soil fertility while simultaneously reducing soil nitrate-N residues and the apparent surplus of N in rain-fed drought-prone regions.