Based on traditional pharmacological applications and partial in vitro data,Cynanchum atratum(CA) is proposed to act on skin whitening. However, its functional evaluation and underlying mechanisms have yet to be identified. This study aimed to examine the anti-melanogenesis activity of CA fraction B (CAFB) on UVB-induced skin hyperpigmentation. Forty C57BL/6j mice were exposed to UVB (100 mJ/cm2, five times/week) for eight weeks. After irradiation, CAFB was applied to the left ear once a day for 8 weeks (the right ear served as an internal control). The results showed that CAFB significantly reduced melanin production in the ear skin, as indicated by the gray value and Mexameter melanin index. In addition, CAFB treatment notably decreased melanin production in α-MSH-stimulated B16F10 melanocytes, along with a significant reduction in tyrosinase activity. Cellular cAMP (cyclic adenosine monophosphate), MITF (microphthalmia-associated transcription factor), and tyrosinase-related protein 1 (TRP1) were also noticeably downregulated by CAFB. In conclusion, CAFB is a promising ingredient for treating skin disorders caused by the overproduction of melanin and its underlying mechanisms involving the modulation of tyrosinase, mainly mediated by the regulation of the cAMP cascade and MITF pathway.