Peripheral nerve injury (PNI) affects millions worldwide, yet currently approved nerve guidance conduits (NGCs) have limited ability to recapitulate the native fascicular architecture that supports organized axonal regeneration. Here, we present an acellular biomimetic multi-fascicle NGC (MFNGC) constructed by integrating electrospun poly(L-lactide-co-ε-caprolactone) (PLCL) membranes with visible-light-crosslinked gelatin hydrogel guide structures, without cells or exogenous growth factors. The MFNGC incorporates three inner conduits mimicking perineurial fascicles enclosed within an epineurium-like outer conduit, approximating key architectural features of the native median nerve in rhesus macaques. In a 12-month exploratory follow-up study using a 15-mm median nerve defect model in three rhesus macaques (Macaca mulatta), the MFNGC showed evidence of functional and histological nerve regeneration, as assessed by compound muscle action potential (CMAP), hand dexterity using pellet clearance test, motor dysfunction scoring, thenar muscle preservation, and axonal remyelination. Nerves regenerated within the MFNGC exhibited organized multi-fascicular tissue formation resembling native nerve architecture, and the conduit showed substantial in vivo degradation, as indicated by a 94% reduction in PLCL molecular weight without obvious chronic inflammatory response at the 12-month endpoint. Although limited by the small number of NHP, this study provides exploratory preclinical evidence supporting further evaluation of an acellular polyfascicular NGC for peripheral nerve regeneration.