Osteogenic differentiation is highly correlated with cell morphology. Morphological changes are a stimulus as well as a consequence of the differentiation process. Besides, geometrical, biochemical and mechanical properties of a substrate can modulate cell adhesion and morphology. Therefore, in the current study, nanofibrous substrate properties were used to implement necessary changes in cell morphology which induced osteogenic differentiation without biological supplements. A polycaprolactone–chitosan nanofiber substrate had been fabricated with an average diameter of ∼75 nm and an appropriate ratio of polymers that balances surface biocompatibility as well as mechanical strength. DSC and wide-angle XRD analysis revealed miscibility between polymers; whereas a degradation study confirmed the structural integrity of nanofibers. Nanofibers did not cause any cytotoxicity to MC3T3-E1 cells as confirmed by Live/Dead® staining. Morphological studies by SEM and confocal microscopy showed significant changes in terms of cell shape, area, compactness, aspect ratio and nucleus area in cells grown on nanofibers which indicated the osteogenic differentiation inducing potential of nanofibers. This was further confirmed by enhanced mineral deposition and alkaline phosphatase activity up to three weeks. In summary, polycaprolactone–chitosan nanofibers could induce early osteogenic differentiation in MC3T3-E1 pre-osteoblasts without any biological supplements by modulating cell morphology. Moreover, cell morphological features can be used as a predictive and informative approach at the early stages of differentiation experiments.