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Background: Melanoma is a highly aggressive skin cancer with limited therapeutic response. Targeting intracellular signaling pathways and promoting tumor cell differentiation are promising therapeutic strategies. Pentoxifylline (PTX) and norcantharidin (NCTD) have demonstrated antitumor properties, but their combined mechanisms of action in melanoma remain poorly understood. Methods: The effects of PTX (30 and 60 mg/kg) and NCTD (0.75 and 3 mg/kg), administered alone or in combination, in a DBA/2J murine B16-F1 melanoma model via intraperitoneal and intratumoral (IT) routes were evaluated. Tumor growth was monitored, and molecular analyses included RNA sequencing, immunofluorescence quantification of PI3K, AKT1, mTOR, ERBB2, BRAF, and MITF protein levels, and molecular docking simulations were performed. Results: Combination therapy significantly reduced tumor volume compared to monotherapies, with relative tumor volume decreasing from 18.1 in the IT control group to 0.6 in the IT combination-treated group. RNA-seq revealed over 3,000 differentially expressed genes in intratumoral treatments, with enrichment in pathways related to oxidative stress, immune response, and translation regulation (KEGG and Reactome analyses). Minimal transcript-level changes were observed for BRAF and PI3K/AKT/mTOR genes; however, immunofluorescence showed reduced total and phosphorylated levels of PI3K, AKT1, mTOR, BRAF, and ERBB2. MITF protein levels and pigmentation increased, especially in PTX-treated groups, indicating enhanced melanocytic differentiation. Docking analyses predicted direct binding of both drugs to PI3K, AKT1, mTOR, and BRAF, with affinities ranging from -5.7 to -7.4 kcal/mol. Conclusions: The combination of PTX and NCTD suppresses melanoma progression through dual mechanisms: inhibition of PI3K/AKT/mTOR signaling and promotion of tumor cell differentiation.