Since the current treatments have not resulted in the desired outcomes for melanoma patients, there is a need to identify more effective medications. Together with other snake venom proteins, cytotoxin-II has shown promising results in tumoral cells. In this study, recombinant cytotoxin-II (rCTII) was expressed in SHuffle® T7 Express cells, while the epitope mapping of rCTII was performed to reveal the antibody-binding regions of rCTII. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to assess the viability of SK-MEL-3 and HFF-2 cells after treating these cells with rCTII. The qRT-PCR was performed to evaluate the expression levels of matrix metallopeptidase 3 (MMP-3), SMAD2, SMAD3, caspase-8, caspase-9, and miR-214 in order to reveal the rCTII-induced signaling pathways in melanoma. Our results have shown that two regions of amino acids, 6-16 and 19-44, as predicted epitopes of this toxin, are essential for understanding the toxicity of rCTII. Treating the melanoma cells with rCTII substantially inhibited the transforming growth factor-beta (TGF-β)-SMAD signaling pathway and down-regulated the expression of MMP-3 and miR-214 as well. This cytotoxin also restored apoptosis mainly via the intrinsic pathway. The down-regulation of MMP-3 and miR-214 might be associated with the anti-metastatic property of rCTII in melanoma. The inhibitory effect of rCTII on the TGF-β signaling pathway might be associated with increased apoptosis and decreased cancer cell proliferation. It is interesting to see that the IC50 value of rCTII has been lower in the melanoma cells than non-tumoral cells, which may indicate its potential effects as a drug. In conclusion, rCTII, as a novel medication, might serve as a potent and efficient anticancer drug in melanoma.