Backgrounds: Polymyxin B， which is a last line antibiotic for multi-drug resistant gram-negative bacteria, has been reported to induce skin hyperpigmentation in patients during treatments. 8~15% or more cases were reported hyperpigmentation, especially in head and neck, seriously influencing the compliance of patients. To investigate the mechanism of hyperpigmentation is beneficial for intervention strategy and improve compliance. Methods: The melanoma cells, SK-MEL-2 cells were employed in the present study to verify whether polymyxin B treatment could directly induce hyperpigmentation. Melanin contents were measured by hot alkali lysis method and tyrosinase were quantified by dopaquinone oxidation method. The real-time quantitative PCR was applied to measure the mRNA levels of melanogenesis-related genes to investigate the possible signaling pathway involved in the polymyxin B induced hyperpigmentation. Results: The melanin content and tyrosinase activity were up-regulated after 5 μg/mL polymyxin B treatment in SK-MEL-2 cells at 48 hr. and 72 hr. The mRNA levels of melanogenesis-related genes were up-regulated, including PI3K, Akt, GSK-3β, CREB, MITF in the polymyxin B treatment group compared with control group at 48 hr. (p<0.001). Conclusions: In the present study, for the first time to reveal that polymyxin B induced hyperpigmentation involved the signaling pathway of PI3K/AKT/GSK-3β and the up-regulation of CREB and MITF subsequently, then induced the activity of tyrosinase and melanin content in melanoma cells.