Adipose-derived stem cell (ASC)-based therapy is a promising treatment strategy for diseases of the musculoskeletal system, as ASCs have the potential to differentiate into numerous cell lineages. However, this field has only recently been explored; therefore, a considerable amount of work is required to determine the therapeutic potential of ASCs. The mechanisms and factors associated with ASC proliferation and differentiation remain to be elucidated. In order to determine the biological properties and subsequent clinical applications of ASCs, these molecular mechanisms must be investigated. The transcriptional co-activator yes-associated protein (YAP), which is a major target of the Hippo signaling pathway, has been reported to serve a crucial role in stem cell proliferation and differentiation. To the best of our knowledge, the role of YAP in the proliferation and differentiation of rat ASCs (rASCs) has not yet been reported. The results of an immunofluorescence analysis revealed that subcellular distribution of YAP in rASCs was regulated by cell density and the actin cytoskeleton. Furthermore, western blot analysis demonstrated that YAP protein expression in rASCs was regulated by lysophosphatidic acid and the actin cytoskeleton. In addition, YAP activation promoted the proliferation of rASCs, whereas YAP inactivation promoted osteogenesis and inhibited adipogenesis of rASCs. In conclusion, these findings demonstrated that YAP may regulate the proliferation and differentiation of rASCs. Targeted modulation of YAP in rASCs may therefore increase the therapeutic effect of rASCs in musculoskeletal diseases.