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As one of the most abundant interstitial elements, nitrogen (N) is effective in improving yield strength of metallic materials, due to interstitial solid solution strengthening. Doping N can substantially enhance the yield strength but often leads to a decreased ductility, revealing a strength-ductility trade-off phenomenon. Here, we simultaneously enhance the strength and ductility in a non-equiatomic Cr-Mn-Fe-Co-Ni high-entropy alloy via N alloying and unravel the underlying microscopic mechanisms. The N-doped alloy (1 at.% N) shows an excellent combination of higher yield strength (104% increase) and larger ductility (38% increase), with a two-stage strain hardening behavior, compared to the N-free alloy. Detailed transmission electron microscopy (TEM) analysis reveals that N-doping introduces more short-range order (SRO) domains within the microstructure, leads to pronounced planar slip, and promotes the formation of nano-spaced (6–15 nm) stacking faults and deformation twins. Continuous generation and interaction of the fine-spaced SFs act as a strong barrier for dislocation movement and provide ample room for dislocation storage. The interaction of SRO with dislocations and the evolution of SFs ascribe to the first strain hardening stage, and the disordering of the SRO along with the activation of deformation twins are attributed to the second strain hardening stage. Our work shows that N-doping is effective in simultaneously improving the strength-ductility synergy and provides novel insights into alloy design with slightly elevating the SFE, and manipulating the ordered structure within the HEA.