Objective: Evaluation of extracellular matrics (ECMs) effect on differentiation of embryonic stem cells (ESCs) to pancreatic β-cell. Materials and Methods: Mouse ESC line, Royan B1, was subjected to differentiation into β-like cells in a three-step method: generation of embryoid bodies (EBs), spontaneous differentiation and induction by Nicotinamide onto different matrices including poly L-ornithine/laminin, gelatin, and two different dilution of matrigel (1:30, 1:100) and control group (no ECM). At the final step, differentiated cells were analyzed for expression of some pancreas-specific genes using "semi-quantitative RT-PCR ", for detection of insulin and C-peptide presence in cells using "immunocytochemistry" and for the evaluation of the amount of secretd insulin in response to glucose Using "insulin secretion assay". Results: The semi-quantitative RT-PCR analysis of differentiated cells on 1:30 matrigel coated-plates showed consistent higher expression of β-cell specific markers including Insulin I, Insulin II, Slc2a2 in comparison to the other ECMs. The results of immunostainig for C-peptide showed no significant differences between the experimental groups and finally insulin secretion assay revealed that differentiated cells on 1:30 matrigel coated-plates secreted more insulin in response to glucose in comparison to the other ECMs. Conclusion: Our results suggest that type of ECM may influence ESC differentiation into insulin-secreting cells and 1:30 matrigel was more effective. However, the success rate of differentiation needs further investigations using other appropriate ECMs.