Melanoma is a type of aggressive cancer. Recent studies have indicated that blue light has an inhibition effect on melanoma cells, but the effect of photobiomodulation (PBM) parameters on the treatment of melanoma remains unknown. Thus, this study was aimed to investigate B16F10 melanoma cells responses to PBM with varying irradiance and doses, and further explored the molecular mechanism of PBM. Our results suggested that the responses of B16F10 melanoma cells to PBM with varying irradiance and dose were different and the inhibition of blue light on cells under high irradiance was better than low irradiance at a constant total dose (0.04, 0.07, 0.15, 0.22, 0.30, 0.37, 0.45, 0.56 or 1.12 J/cm), presumably due to that high irradiance can produce more ROS, thus disrupting mitochondrial function.