Introduction: Different effects of low level laser irradiation (LLLI) on various cell types have already been demonstrated. However, its effects on embryonic stem cells have not yet been shown. The present study evaluates the morphological and immunocytochemical effects of LLLI on human embryonic stem cell (hESC) colonies. Material and Methods: Equal-sized pieces of hESC line (Royan H1) were irradiated with a single dose of 830-nm Ga-Al-As diode laser (3, 5, and 8 jcm-2, 30mW) and cultured on mouse embryonic fibroblasts. The morphology of the colonies was evaluated qualitatively by observation under an inverted microscope (grades A, B, C, and D exhibited 0-30%, 30-50%, 50-80%, and 80-100% differentiation, respectively). The stemness area was assessed by expression of surface antigens using anti-Tra-1-60 and anti-Tra-1-81. Results: Our data demonstrated a dose-dependent stimulatory effect of LLLI on hESC differentiation. Two doses of 5 and 8jcm-2 induced statistically significant differentiation (grades C and D). Conclusions: These data showed that LLLI influenced hESC differentiation, which might be used for cell therapy after transplantation.