<p>Long-segment peripheral nerve injury (PNI) often results in persistent functional impairment due to the limited capacity of regeneration and poor functional recovery. Nerve conduits have emerged as a promising strategy to enhance regeneration, particularly those that modulate the immune microenvironments and direct macrophage polarization. In this study, an advanced nerve conduit composed of an outer tube of aligned electrospun poly(lactic-<i>co</i>-glycolic acid) fiber and an inner chiral poly(amino acid) hydrogel is designed to promote PNI repair by inducing macrophage polarization toward the M2 phenotype. The chiral hydrogels are prepared by combining poly(<span>l</span>-lysine) or poly(<span>d</span>-lysine) with poly(<span>l</span>-glutamic acid), resulting in P_L-Lys+P_L-Glu and P_D-Lys+P_L-Glu formulations, respectively. Both hydrogels increase the M2/M1 macrophage ratio by 0.85 and 1.03, respectively, and significantly improve motor function recovery and axonal remyelination in a rat model with sciatic nerve defect. Notably, the aligned fiber conduit filled with P_D-Lys+P_L-Glu achieves a compound muscle action potential of (19.82 ± 1.30) mV, a sciatic functional index of (−55.47 ± 3.56), and a myelin sheath thickness of (0.96 ± 0.12) μm—comparable to those observed in the Autograft group. Transcriptomic analysis reveals that the chiral hydrogel enhances recognition of extracellular matrices and promotes focal adhesion formation, thereby facilitating myelination by activating the PI3K/Akt signaling pathway. In summary, the aligned fiber nerve conduit containing chiral poly(amino acid) hydrogel shows strong potential for immunomodulation and functional recovery in the treatment of long-segment PNI.</p> Graphical abstract <p></p>