Traditional Chinese medicine (TCM) is a rich resource of anticancer drugs. Increasing bioactive natural compounds extracted from TCMs are known to exert significant antitumor effects, but the action mechanisms of TCMs are far from clear. Proteomics, a powerful platform to comprehensively profile drug-regulated proteins, has been widely applied to the mechanistic investigation of TCMs and the identification of drug targets. In this paper, we discuss several bioactive TCM products including terpenoids, flavonoids, and glycosides that were extensively investigated by proteomics to illustrate their antitumor mechanisms in various cancers. Interestingly, many of these natural compounds isolated from TCMs mostly exert their tumor-suppressing functions by specifically targeting mitochondria in cancer cells. These TCM components induce the loss of mitochondrial membrane potential, the release of cytochrome c, and the accumulation of ROS, initiating apoptosis cascade signaling. Proteomics provides systematic views that help to understand the molecular mechanisms of the TCM in tumor cells; it bears the inherent limitations in uncovering the drug-protein interactions, however. Subcellular fractionation may be coupled with proteomics to capture and identify target proteins in mitochondria-enriched lysates. Furthermore, translating mRNA analysis, a new technology profiling the drug-regulated genes in translatome level, may be integrated into the systematic investigation, revealing global information valuable for understanding the action mechanism of TCMs.