Melanoma, the most serious type of skin cancer, exhibits a high risk of metastasis. Although chemotherapeutic treatment for metastatic melanoma improves disease outcome and patient survival, some patients exhibit resistance or toxicity to the drug treatment regime. OTUB1 is a deubiquitinating enzyme overexpressed in several cancers. In this study, we investigated the effects of inhibiting OTUB1 expression on melanoma cell proliferation and viability and identified the underlying molecular mechanism of action of OTUB1. Endogenous OTUB1 knockdown in melanoma cells was performed using short interfering RNA, and the resulting phenotypes were assessed via MTT assays, Western blotting, and cell cycle analysis. Differentially expressed genes between OTUB1-knockdown cells and control cells were identified using RNA sequencing and confirmed via Western blotting and reverse transcription polymerase chain reaction. Furthermore, the involvement of apoptotic and cell survival signaling pathways upon OTUB1 depletion was investigated. OTUB1 depletion in melanoma cells decreased cell viability and caused simultaneous accumulation of cells in the sub-G1 phase, indicating an increase in the apoptotic cell population. RNA sequencing of OTUB1-knockdown cells revealed an increase in the levels of the apoptosis-inducing protein TRAIL. Additionally, OTUB1-knockdown cells exhibited increased sensitivity to PLX4032, a BRAF inhibitor, implying that OTUB1 and BRAF act collectively in regulating apoptosis. Taken together, our findings show that OTUB1 induces apoptosis of melanoma cells in vitro, likely through the upregulation of TRAIL, and suggest that approaches targeting OTUB1 can be developed to provide novel therapeutic strategies for treating melanoma.