Mesenchymal stromal cells (MSCs) hold great promise for regenerative medicine. Stable ex vivo gene transfer to MSCs could improve the outcome and scope of MSC therapy, but current vectors require multiple rounds of transduction, involve genotoxic viral promoters and/or the addition of cytotoxic cationic polymers in order to achieve efficient transduction. We describe a self-inactivating foamy virus vector (FVV), incorporating the simian macaque foamy virus envelope and using physiological promoters, which efficiently transduces murine MSCs (mMSCs) in a single-round. High and sustained expression of the transgene, whether GFP or the lysosomal enzyme, arylsulphatase A (ARSA), was achieved. Defining MSC characteristics (surface marker expression and differentiation potential), as well as long-term engraftment and distribution in the murine brain following intracerebroventricular delivery, are unaffected by FVV transduction. Similarly, greater than 95% of human MSCs (hMSCs) were stably transduced using the same vector, facilitating human application. This work describes the best stable gene transfer vector available for mMSCs and hMSCs.