Despite decades of study, the antiproliferation and molecular processes of the biomolecular elements of honeybee venom (i.e., melittin) as anticancer agents remain largely unclear. This study illustrated the antiproliferation effect of melittin by regulating IL-1β, COX-2 and TNF-α in human colorectal cancer cells WiDr. In order to assess the antiproliferation, various concentrations of melittin were contacted to the WiDr cell through MTT assay. Moreover, the ability of melittin to regulate the expression of IL-1β, COX-2 and TNF-α was determined by ELISA. The cytotoxic effect showed the inhibition of the WiDr cell in a dose-dependent manner, with the IC10, IC25 and IC50 values of 0.095 ± 0.001, 0.147 ± 0.002 and 0.207 ± 0.004, respectively. Additionally, in further investigations, melittin markedly inhibited the IL-1β and COX-2 expression but induced the TNF-α, demonstrating significant (p < 0.05) elevations compared to the control group. These findings point to the antiproliferative properties of melittin in WiDr cells. Therefore, as a unique natural treatment for colorectal cancer, melittin may have a lot of potential. HIGHLIGHTS In this study, we confirmed melittin has very strong anticancer activity against colorectal cell cancer cells with IC50 values of 0.207 ± 0.004 µM Melittin regulates colorectal cell cancer by inhibiting the IL-1β and COX-2 expression Melittin increases the expression of TNF-α to inhibit colorectal cell cancer GRAPHICAL ABSTRACT