Background Toll-like receptors (TLRs) are expressed on human melanocytes, and play an important role in innate and acquired immunity. The role of TLRs in the pathogenesis of vitiligo has not been fully described. Aim To investigate the expression of TLRs in melanocytes from patients with vitiligo and healthy controls (HCs). Methods Primary cultured vitiligo and control melanocytes were obtained from perilesional normal skin of patients with generalized vitiligo and HCs. TLRs mRNA expression in melanocytes were determined by real-time reverse transcription PCR and protein expression by western blotting. Apoptosis was analysed using an annexin V-fluorescein isothiocyanate apoptosis detection kit, and tyrosinase activity and melanin content were measured by a modified dopachrome and colorimetric method. Interleukin (IL)-6, IL-8 and soluble cell adhesion molecule (sICAM)-1 expression were measured by ELISA. Results In vitiligo melanocytes, compared with control melanocytes, apoptosis rate, expression of TLR7 and TLR9 mRNA and protein, and production of IL-8, IL-6 and sICAM-1 were significantly increased, whereas tyrosinase activity and melanin content were significantly decreased. Conclusions Our results suggest that the increased expression of TLR7 and TLR9 might correlate with melanocyte dysfunction in vitiligo.