The CRISPR-Cas9 system has significantly advanced regenerative medicine research by enabling genome editing in stem cells. Mesenchymal stem cells (MSCs) have recently emerged as highly promising therapeutic agents due to their desirable properties including differentiation ability and cytokine production. While CRISPR-Cas9 technology is being applied to develop MSC-based therapeutics, MSCs exhibit inefficient genome editing and susceptibility to plasmid DNA. In this study, we compared and optimized plasmid DNA and RNP approaches for efficient genome engineering in MSCs. The RNP-mediated approach enabled genome editing with high indel frequency and low cytotoxicity in MSCs. By utilizing Cas9 RNPs, we successfully generated B2M-knockout MSCs, which reduced T cell differentiation and improved MSC survival. Furthermore, this approach enhanced the immunomodulatory effect of IFN-r priming. These findings indicate that RNP-mediated engineering of MSC genomes can achieve high efficiency, and engineered MSCs can potentially be a promising therapeutic strategy.