Considerable research has been focused on the utilization of electrospun ultrafine fibers to construct biomimetic scaffolds for tissue engineering applications. However, there is still a limited understanding of the effects of fiber alignment on stem cells-fibrous scaffold interactions and their role in deciding the fate of stem cells and scaffolds. Here, we tracked the real-time interactions between mesenchymal stem cells (MSCs) and aligned/non-aligned electrospun PCL-gelatin ultrafine fibers using time-lapse microscopy, and investigated the effects of fiber alignment on the behaviour of cell spreading, migration and differentiation, and scaffold remodeling. Cells were found to spread and migrate with increased velocity in the direction of aligned fibers as compared to non-aligned ones. Moreover, enhanced interactions between cells and aligned fibers were found to facilitate the infiltration of cells into the interior of fibrous constructs, which further led to enhanced micro-integration and scaffold remodeling as well as increased gene expression of late osteogenic markers as compared to non-aligned fibrous constructs. This study advances our understanding of the effects of fiber alignment on stem cells-fibrous scaffold interactions, and will facilitate the design of fibrous scaffolds with enhanced cell-matrix interactions for tissue engineering applications.