The effects of extracts of Allium sativum, dipeptide-2 and acetyl hexapeptide-8 on B16F10 cells viability were determined using MTT method. And the effects of these three substances on the melanin synthesis and morphology of B16F10 cells were evaluated with the nontoxic dose. With the single factor experiment, orthogonal experiments were used to explore the concentration of extracts of A. sativum, dipeptide-2 and acetyl hexapeptide-8 on the inhibitory effects of the melanin synthesis. The expression of proteins of TYR、TRP-1 and TRP-2 in B16F10 cells were detected using western blot. The results showed that the extracts of A. sativum(0.00-2.50 μg/mL), dipeptide-2 and acetyl hexapeptide-8 (0.00-4.00 μg/mL) had no effect on the cell viability and morphology of B16F10 cells. The extracts of A. sativum(0.00-2.50 μg/mL), dipeptide-2 and acetyl hexapeptide-8(0.00-4.00 μg/mL) could inhibit the melanogenesis in B16F10 cells with a dose-dependent manner. The optimal inhibition effect on melanogenesis was extracts of A. sativum 2.50 μg/mL, dipeptide-2 1.00 μg/mL, and acetyl hexapeptide-8 0.50 μg/mL. The optimal composition could significantly inhibit the expression of TYR, TRP-1 and TRP-2 proteins, indicating that the inhibition of melanogenesis by the composition was linked to the reduction of TYR, TRP-1 and TRP-2 protein levels.