The present study was conducted to investigate the effects of deep application of nitrogen (N) fertilizer and straw incorporation on grain yield and quality, nutrient accumulation, and root morphological and physiological traits in both rice (Oryza sativa L.) cultivars during 2019 and 2020. The experimental treatments were comprised of deep N fertilization with (DF+S) and without straw incorporation (DF) as well as broadcasting fertilization with (BF+S) and without straw return (BF). No fertilizer and no straw return treatment were taken as control. Results depicted that the DF+S and DF treatment significantly increased the grain yield by 46.4 and 42.2%, respectively, compared with BF. The DF+S also significantly increased total N accumulation by 37.9 and 44.0%, total phosphorus accumulation by 39.3 and 41.7%, total potassium accumulation by 63.6 and 73.3% for the cultivars Yuxiangyouzahn (YXYZ) and Wufengyou615 at maturity, respectively, compared with BF. Moreover, nutrient accumulation and grain yield of BF+S were slightly higher than BF. Straw incorporation resulted in better total root volume and total root length than no straw incorporation treatments in YXYZ, regardless of fertilizer application method. Root morphology traits are positively associated with other positive traits such as yield or nutrient uptake. The DF+S treatment also significantly increased the milled rice rate and remarkably reduced the chalkiness degree for both rice cultivars, compared to BF treatment. Overall, DF+S is recognized as the optimal method to improve rice productivity and quality with better nutrient acquisition.