Melanin, an important virulence factor of pathogenic fungi, has been shown to suppress host immune responses in multiple ways. Autophagy is a vital cellular mechanism underlying the host’s innate immunity against microbial infections. However, the potential influence of melanin on autophagy has not been explored. We investigated the effect of melanin on autophagy in macrophages, which play a key role in controllingSporothrixspp. infection, as well as the mechanism of melanin interaction with Toll-like receptor (TLR)-induced pathways.Sporothrix globosaconidia (wild-type and melanin-deficient mutant strains) or yeast cells were co-cultured with THP-1 macrophages to demonstrate that, althoughS.globosainfection led to the activation of autophagy-related proteins and increased autophagic flux,S.globosamelanin suppressed macrophage autophagy. Incubation withS.globosaconidia also increased the expression levels of reactive oxygen species and multiple proinflammatory cytokines (interleukin-6, tumor necrosis factor-α, interleukin-1β and interferon-γ) in macrophages. These effects were attenuated as melanin presented. Furthermore, whileS.globosaconidia significantly increased the expression of both TLR2 and TLR4 in macrophages, the knockdown of TLR2, but not TLR4, with small interfering RNA suppressed autophagy. Overall, this study revealed the novel immune defense ability ofS.globosamelanin to inhibit macrophage functionality by resisting macrophage autophagy through the regulation of TLR2 expression.