Nitrogen is a major driving force for the improvement of crop yield worldwide,but brings detrimental effects on ecosystems.Therefore, enhancing nitrogen use efficiency (NUE)is vital forsustainableagriculture. The nitrate transporter (NRT/NPF) family associated with nitrogen uptake and utilization isindispensable to the improvement of NUE in crops. As an important food security crop, cassava (Manihot esculenta) produces an acceptable yield in nutrient‐deficient soil. Here we identified and systematically analyzed the NPF gene family in cassava, including phylogenetic relationship, chromosome location, gene duplication, and gene expression in response to different nitrogen supply. The stem is involved in nitrogen transportation and remobilization. Gene expression analysis revealed thatMeNPF5.4 and MeNPF6.2 were specifically expressed in stem, andhave diverse expression in different nitrogen conditions.To facilitate the functional analysis of MeNPF5.4 and MeNPF6.2, we constructed their overexpression (OE) lines in rice. A NO3− flux assayshowed that MeNPF5.4and MeNPF6.2OE lines exhibited a marked decrease NO3− efflux and significant NO3− influxcompared with WT, which suggests that they might have contributed to NUE improvementof rice. Notably, overexpressing MeNPF5.4showed increased grain size, grain number and weight per panicle. More importantly,MeNPF5.4 OE linecontributed to salt tolerance. Nevertheless, anobvious reduction in grain number and weight per panicle was detected in the MeNPF6.2 OE line compared with WT. Strikingly, MeNPF6.2OE line showed higher salt stress tolerance than WT in LN conditions. Taken together, our results demonstrated that MeNPF5.4 can potentially improve the NUE and salt stress tolerance of rice,which reveals valuable breeding targets to improve crop yield and stress tolerance.This article is protected by copyright. All rights reserved