The TLR4/NF-κB signaling pathway plays a critical role in tumor progression. Andrographolide (Andro) has been reported to have anticancer activity in multiple types of cancer. However, the pharmacological activities of Andro in melanoma are not completely understood. In this study, we defined the anticancer effects of Andro in melanoma and elucidated its potential mechanisms of action. Our experiments showed that Andro significantly inhibited melanoma tumor growth and metastasis by inducing cell cycle arrest and apoptosis. In addition, Andro significantly inhibited the TLR4/NF-κB signaling pathway. Furthermore, the inactivation of TLR4/NF-κB signaling inhibited the mRNA and protein expression of CXCR4 and Bcl-6, which are antitumor genes. This work provides evidence that the TLR4/NF-κB signaling pathway is a potential therapeutic target and may also be indispensable in the Andro-mediated anticancer effect in melanoma.