Although the toxicogenomics of kojic acid treated A375 human malignant melanoma cells has been elucidated, the proteomics of cellular response is still poorly understood. We performed proteomic analysis to investigate the anticancer effect of kojic acid on protein expression profile in A375 cells. A375 cells were treated with kojic acid at 8 microg/mL for 24, 48, and 72 h. With the use of 2-D PAGE and MALDI-Q-TOF MS and MS/MS analyses, proteomic profiles of A375 cells between control and kojic acid treatment were compared, and 30 differentially expressed proteins, containing 2 up-regulated proteins and 28 down-regulated proteins, were identified. Among these proteins, 17 isoforms of 5 identical proteins were observed and 11 chaperone proteins showed the high proportion of protein spots with 36.7% of total proteins. Bioinformatic tools were used to search for protein function and prediction of protein interaction. Sixteen differentially expressed proteins exhibited interaction network linked to the downstream regulations of p53 tumor suppressor and cell apoptosis, which may lead to suppress the melanogenesis and tumorigenesis of kojic acid treated A375 cells. In addition, GRP75, VIME and 2AAA were validated by Western blot analysis, whereas GRP75, 2AAA, HS90B, ENPL and KPYM were validated by RT-PCR. Therefore, these proteins play the important roles in cancer progression and may be potential biomarkers that are useful for diagnostic and therapeutic applications of malignant melanoma cancer.