Induced pluripotent stem cells (iPSCs) have been directly generated from fibroblast cultures though retrovirus- or lentivirus-mediated ectopic overexpression of only a few defined transcriptional factors. This remarkable achievement has greatly enhanced our ability to explore the causes of, and potential cures for, many genetic diseases, and strengthened the promise of regenerative medicine. In fact, to date, many kinds of somatic cells from different tissues have exhibited a capacity for reprogramming toward an embryonic stem cell-like state, but major bottlenecks in iPSC derivation and therapeutic use remain, including low reprogramming efficiencies and the tumorigenesis of the generated iPSC. Here, we successfully generated miR-302s-induced pluripotent stem cells (mirPS cells) from human embryonic kidney (HEK) 293 cells via transfection of the miR-302s expression vector. We also determined the optimal culture conditions to generate mirPS on feeder cells, which included the use of serum-free N2B27 medium. The mirPS cells generated by our improved conditions showed the expression of pluripotent marker genes such as OCT3/4, NANOG, and SOX2 under growth conditions via reverse transcription-PCR, whereas no expression of these genes was observed in HEK293 cells. On the other hand, under differentiation conditions, mirPS cells formed ball-shaped structures (embryoid bodies), and showed the ability to differentiate into three germ layers (ectoderm, mesoderm, and endoderm) in vitro. The results suggested that our generated mirPS cells are actually functional as a cell resource to apply to regenerative medicine, and mirPS cells are suitable materials to clarify the mechanism underlying the reprogramming from somatic cells.