Tissue regeneration is a complex process in which numerous chemical and physical signals are coordinated in a specific spatiotemporal pattern. In this study, we tested our hypothesis that cell migration and bone tissue formation can be guided and facilitated by microscale morphological cues presented from a scaffold. We prepared poly(l-lactic acid) (PLLA) electrospun fibers with random and aligned structures and investigated their effect on in vitro migration of human mesenchymal stem cells (hMSCs) and in vivo bone growth using a critical-sized defect model. Using a polydopamine coating on the fibers, we compared the synergistic effects of chemical signals. The adhesion morphology of hMSCs was consistent with the direction of fiber alignment, whereas the proliferation of hMSCs was not affected. The orientation of fibers profoundly affected cell migration, in which hMSCs cultured on aligned fibers migrated 10.46-fold faster along the parallel direction than along the perpendicular direction on polydopamine-coated PLLA nanofibers. We implanted each fiber type into a mouse calvarial defect model for 2 months. The micro-computed tomography (CT) imaging demonstrated that regenerated bone area was the highest when mice were implanted with aligned fibers with polydopamine coating, indicating a positive synergistic effect on bone regeneration. More importantly, scanning electron microscopy microphotographs revealed that the direction of regenerated bone tissue appeared to be consistent with the direction of the implanted fibers, and transmission electron microscopy images showed that the orientation of collagen fibrils appeared to be overlapped along the direction of nanofibers. Taken together, our results demonstrate that the aligned nanofibers can provide spatial guidance for in vitro cell migration as well as in vivo bone regeneration, which may be incorporated as major instructive cues for the stimulation of tissue regeneration.