In this study, we offer new insights into the contrasting effects of electrospun fiber orientation on microglial polarization under normoxia and hypoxia, and establish for the first time, the intrinsically protective roles of electrospun meshes against hypoxia-induced microglial responses. First, resting microglia were cultured under normoxia on poly(caprolactone) fibers possessing two distinctly different fiber orientations. Matrix-guided differences in cell shape/orientation and differentially expressed Rho GTPases (RhoA, Rac1, Cdc42) were well-correlated with the randomly oriented fibers inducing a pro-inflammatory phenotype and the aligned fibers sustaining a resting phenotype. Upon subsequent hypoxia induction, both sets of meshes offered protection from hypoxia-induced damage by promoting a radical phenotypic switch and beneficially altering the M2/M1 ratio to different extents. Compared to 2D hypoxic controls, meshes significantly suppressed the expression of pro-inflammatory markers (IL-6, TNF-α) and induced drastically higher expression of anti-inflammatory (IL-4, IL-10, VEGF-189) and neuroprotective (Nrf-2) markers. Consistent with this M2 polarization, the expression of Rho GTPases was significantly lower in the mesh groups under hypoxia compared to normoxic culture. Moreover, meshes - particularly with aligned fibers - promoted higher cell viability, suppressed caspase 3/8 and LC-3 expression and promoted LAMP-1 and LAMP-2 expression, which suggested the mitigation of apoptotic/autophagic cell death via a lysosomal membrane-stabilization mechanism. Notably, all protective effects under hypoxia were observed in the absence of additional soluble cues. Our results offer promise for leveraging the intrinsic therapeutic potential of electrospun meshes in degenerative diseases where microglial dysfunction, hypoxia and inflammation are implicated. © 2021 IOP Publishing Ltd.