Human pluripotent stem cells (hPSCs) exist in at least two distinct states in mammals: naïve pluripotency that represents several molecular characteristics in pre-implantation epiblast and primed pluripotency that corresponds to cells poised for differentiation in post-implantation epiblast. To identify and characterize the surface molecules that are necessary for the maintenance of naïve hPSCs, we generated a panel of murine monoclonal antibodies (MAbs) specific to the naïve state of hPSCs. Flow cytometry showed that N1-A4, one of the MAbs, bound to naïve hPSCs but not to primed hPSCs. Cell surface biotinylation and immunoprecipitation analysis identified that N1-A4 recognized heat shock protein 60 (HSP60) expressed on the surface of naïve hPSCs. Quantitative polymerase chain reaction (qPCR) analysis showed that HSP60 expression was rapidly downregulated during the embryoid body (EB) differentiation of primed hPSCs. HSP60 knockdown led to a decrease in the expression of pluripotency genes in primed hPSCs. HSP60 depletion also led to a decrease in the expression of pluripotency genes and representative naïve-state-specific genes in naïve hPSCs. Taken together, the results suggest that HSP60 is downregulated during differentiation of hPSCs and is required for the maintenance of pluripotency genes in both primed and naïve hPSCs, suggesting that HSP60 is a regulator of hPSC pluripotency and differentiation.