Abnormal melanin overproduction leads to hyperpigmentation disorders, and tyrosinase is the rate-limiting enzyme in melanin biosynthesis. To develop natural and safe tyrosinase inhibitors, this study systematically explored anti-melanogenic peptides from the muscle proteins of Takifugu obscurus. Five commercial proteases (pepsin, trypsin, flavourzyme, papain, and alkaline protease) were used for enzymatic hydrolysis, and pepsin was identified as the optimal enzyme. The pepsin-hydrolyzed peptide fraction (300–1000 Da) exhibited a tyrosinase inhibition rate of 87.01% ± 2.64% at 10 mg/mL, comparable to arbutin (96.76% ± 1.12%) but with significantly lower cytotoxicity (cell viability > 90% at 4.0 mg/mL vs 56.43% ± 1.20% for arbutin). Peptide identification via nano-LC-MS/MS and virtual screening using molecular docking with mushroom tyrosinase led to the selection of five candidate peptides (Pep1–Pep5). Notably, Pep2 (LRVAPEEHPTL) showed the highest binding affinity and significantly outperformed arbutin in in vitro tyrosinase inhibition at certain concentrations. In α-MSH-stimulated B16-F10 melanoma cells, both crude pepsin hydrolysates and synthetic Pep2 effectively reduced melanin content and cellular tyrosinase activity without obvious cytotoxicity. Furthermore, an in vivo zebrafish model confirmed that the pepsin-hydrolyzed peptides effectively inhibited melanin accumulation while maintaining excellent biosafety (near 100% survival), overcoming the severe toxicity limitations of arbutin. Mechanistic investigation using real-time quantitative reverse transcription PCR and global RNA-seq transcriptomic analysis revealed that Pep2 downregulated key genes involved in melanogenesis, including Mitf, Tyr, Tyrp1, Dct, Mc1r, and Mapk1. These findings demonstrate that T. obscurus peptides, particularly LRVAPEEHPTL, are promising natural tyrosinase inhibitors with a multi-level regulatory network. Alongside direct enzyme binding and classical transcriptional suppression, transcriptomic profiling suggests their potential to modulate the cAMP-PDE axis, further blunting the upstream melanogenic signal.  These synergistic properties offer great potential for applications in functional foods and cosmetic formulations.