The tRNA-derived fragments (tRFs) are small non-coding RNAs containing fewer than 50 nucleotides that area bundant in humans and have various biological functions. However, their roles and mechanisms in melanogenesis are unclear. In this study, we firstly investigated the change in the expression profile of tRFs in melanocytes after UVB irradiation through tsRNA sequencing. UVB-induced melanogenesis led to the upregulation of 119 tRFs and the down regulation of 103 tRFs. Among the tRFs with greater than 1.5-fold change in expression level, tRF-Ser-AGA-002 (tRF-5022a) was significantly upregulated after UVB irradiation. Overexpression of tRF-5022a in melanocytes significantly increased melanin production and upregulated melanogenesis-related genes. Ontheotherhand, inhibitionoftRF-5022aexpressionhadthe opposite effects. CCDC88A, which regulates the phosphatidylinositol 3-kinase (PI3K) /protein kinase B (Akt) signaling pathway, was significantly downregulated in the tRF-5022a-overexpressing melanocytes. Collectively, our data indicates that the expression profile of tRFs was significantly altered in melanocytes after UVB irradiation, and tRF-5022a is involved in regulating melanogenesis.