Human mesenchymal stem cells (hMSCs) have attracted significant attention for tissue engineering because of their ability to differentiate into bone cells, chondrocytes, adipocytes, and muscle cells. Single-walled carbon nanotubes (SWCNTs) have been considered as a potential material for tissue engineering applications due to their unique properties, such as high aspect ratio, excellent electrocatalytic activity, and biocompatibility. Here we prepared exfoliated SWCNTs layers through an ultra-sonication process in the acidic medium and evaluated their cytotoxicity using hMSCs. Improved viability and osteogenesis of hMSCs were observed in the presence of exfoliated SWCNTs. Besides, the higher expression of osteogenic differentiation-related genes in the presence of exfoliated SWCNTs further confirmed their enhanced osteogenic nature. These results indicated the potential of SWCNTs as a biomaterial for tissue engineering applications.