Cells sense mechanical cues and convert them into biochemical responses to regulate biological processes such as embryonic development, aging, cellular homeostasis, and disease progression. In this study, we introduce a large-scale, systematic approach to identify proteins with mechanosensitive nuclear localization, highlighting their potential roles in mechanotransduction. Among the proteins identified, we focus here on the splicing factor PTBP1. We demonstrate that its nuclear abundance is regulated by mechanical cues such as cell density, size, and extracellular matrix (ECM) stiffness and that PTBP1 medicates the mechanosensitive alternative splicing of the endocytic adapter protein Numb. Furthermore, we show that PTBP1 and Numb alternative splicing is critical for ECM stiffness-induced epithelial cell spreading and proliferation as well as for mesenchymal stem cell differentiation into osteoblasts on a stiff matrix. Our results underscore the emerging role of alternative splicing in mechanotransduction and provide novel mechanistic insights into how matrix stiffness modulates cellular mechanoresponses.