Three-dimensional (3D) culture systems have been shown to enhance cellular secretion of small extracellular vesicles (sEVs) compared to two-dimensional (2D) culture. However, the molecular mechanisms driving sEV secretion and influencing their potential for disease treatment have not been elucidated. In this study, we discovered the depolymerisation of cortical actin as a new mechanism that leads to increased sEV release, and that in 3D cultured mesenchymal stem cells (MSCs), this process was modulated by the downregulation of integrin-α1 (ITGA1) and subsequent inhibition of the RhoA/cofilin signalling pathway. Interestingly, the knockdown of Rab27A and Rab27B significantly reduced sEV secretion by MSCs to 0.5- and 0.1-fold, respectively. However, there was no difference in expression levels of Rab27A/B between MSCs cultured in 2D and 3D environments. In addition, sEVs derived from 3D cultured MSCs demonstrated enhanced therapeutic function both in vitro and in rat models of osteoarthritis (OA) and wound healing. Collectively, this study illustrates a new mechanism for enhanced secretion of sEVs, involving RhoA/cofilin pathway-dependent cortical actin depolymerisation, which is independent of Rab27A/B. These findings provide novel insights for optimising the yield of stem cell-derived sEVs, as well as their therapeutic efficacy for treating chronic diseases.