Human embryonic stem cells (hESCs) are thought to be a promising cell source for cell transplantation therapy. For such a clinical application, the hESCs should be manipulated using appropriate and qualified materials. In this study, we examined the efficacy of recombinant human laminin (rhLM) isoforms on the undifferentiated growth of hESCs. We first determined the major integrins expressed on the hESCs to reveal the preference of the hESCs for rhLMs, and found that the hESCs mainly expressed integrin alpha6beta1, which binds predominantly to laminin-111, -332 and -511/-521. When the hESCs were seeded onto rhLMs, the cells indeed adhered markedly to rhLM-332, and to rhLM-511 and rhLM-111 to a lesser extent. The hESCs proliferated on these three rhLMs for several passages while preserving their pluripotency. These results show that rhLM-111, -332, and -511 are good substrates to expand undifferentiated hESCs due to their high affinity to integrin alpha6beta1 expressed on hESCs.