Low-level laser irradiation (LLLI) can enhance stem cell (SC) activity by increasing migration and proliferation. This study investigated the effects of LLLI on proliferation, enzymatic activity, and growth factor production in human umbilical cord mesenchymal SCs (hUC-MSCs) as well as the underlying mechanisms. hUC-MSCs were assigned to a control group (non-irradiation group) and three LLLI treatment groups (635 nm group, 808 nm group, and 635/808 nm group). Laser power density and energy density of 20 mW/cm2 and 12 J/cm2, respectively, were used for each experiment. The proliferation rate was higher in the 635 nm as compared to the other groups. LLLI at 808 nm did not induce cell proliferation. ROS levels in cells exposed to 635, 808, and 635/808 nm radiation were increased by 52.81%, 26.89%, and 21.15%, respectively, relative to the control group. CAT, tGPx, and SOD activity was increased. LLLI at 808 nm increased the levels of IL-1, IL-6, and NFκB but not VEGF. LLLI improved hUC-MSCs function and increased antioxidant activity. Dual-wavelength LLLI had more potent effects on hUC-MSCs than single-wavelength treatment. LLLI has potential applications in the preconditioning of hUC-MSCs in vitro prior to transplantation, which could improve the regenerative capacity of cells.