This study aimed to elucidate the response of B16F10 cells to blue light following infection with an empty adenoviral vector (Adv). Twenty-four hours post-infection, the B16F10 cells underwent phototherapy, and a range of cellular parameters were assessed, including cell viability, melanin content, cell cycle progression, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), apoptosis, necrosis, lactate dehydrogenase (LDH) release, and autophagy. The findings indicated that Adv infection elevated melanin content and autophagy, resulting in G1 phase cell cycle arrest. In comparison to the control group, light-treated, Adv-infected cells exhibited reduced inhibition rates, LDH release, apoptosis, and necrosis, while MMP and ROS levels were elevated. The study concluded that although Adv, as a gene delivery vector in conjunction with light therapy, significantly modulated melanin content and autophagy. Moreover, while melanin's light absorption capacity mitigated MMP reduction, the resultant excitation molecules generated ROS, enhancing oxidative stress.