Generating universal human umbilical mesenchymal stem cells (UMSCs) without immune rejection is desirable for clinical application. Here we developed an innovative strategy using CRISPR/Cas9 to generate B2M‐UMSCs in which human leucocyte antigen (HLA) light chain β2‐microglobulin (B2M) was deleted. The therapeutic potential of B2M‐UMSCs was examined in a mouse ischaemic hindlimb model. We show that B2M‐UMSCs facilitated perfusion recovery and enhanced running capability, without inducing immune rejection. The beneficial effect was mediated by exosomes. Mechanistically, microRNA (miR) sequencing identified miR‐24 as a major component of the exosomes originating from B2M‐UMSCs. We identified Bim as a potential target of miR‐24 through bioinformatics analysis, which was further confirmed by loss‐of‐function and gain‐of‐function approaches. Taken together, our data revealed that knockout of B2M is a convenient and efficient strategy to prevent UMSCs‐induced immune rejection, and it provides a universal clinical‐scale cell source for tissue repair and regeneration without the need for HLA matching in the future.