Kinases are involved in numerous critical cell signaling processes, and dysregulation in kinase signaling is implicated in many types of human cancers. In this study, we applied a parallel-reaction monitoring (PRM)-based targeted proteomic method to assess kinome reprogramming during melanoma metastasis in three pairs of matched primary/metastatic human melanoma cell lines. Around 300 kinases were detected in each pair of cell lines, and the results showed that Janus kinase 3 (JAK3) was with reduced expression in the metastatic lines of all three pairs of melanoma cells. Interrogation of The Cancer Genome Atlas (TCGA) data showed that reduced expression of JAK3 is correlated with poorer prognosis in melanoma patients. Additionally, metastatic human melanoma cells/tissues exhibited diminished levels of JAK3 mRNA relative to primary melanoma cells/tissues. Moreover, JAK3 suppresses the migration and invasion of cultured melanoma cells by modulating the activities of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9). In summary, our targeted kinome profiling method provided by far the most comprehensive dataset for kinome reprogramming associated with melanoma progression, which builds a solid foundation for examining the functions of other kinases in melanoma metastasis. Moreover, our results reveal a role of JAK3 as a potential suppressor for melanoma metastasis.