Flavokawain C (FKC), a naturally occurring chalcone, has previously been shown to inhibit the growth of colon carcinoma HCT 116 cells through induction of apoptosis and cell cycle arrest. However, the possible underlying mechanisms of cell death as a response to FKC treatment remains unclear. In this study, we performed proteomic analysis of HCT 116 cells treated with FKC to identify proteins that change in abundance. This was followed by bioinformatic analysis to predict possible associated molecular targets or pathways involved in the observed effects of FKC. A total of 35 proteins that changed in abundance (17 increased and 18 decreased) were identified through two-dimensional gel electrophoresis followed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Using the Ingenuity Pathway Analysis (IPA), these proteins were predicted to be involved in cell death and survival, cell cycle, cellular growth and proliferation, protein synthesis, post-translational modification and amino acid metabolism by. Further analysis of the transcript levels of selected proteins using qPCR showed that some of the genes exhibited similar change of profile to that of the proteins'. Our results have provided novel insights into the potential molecular mechanisms underlying FKC-induced apoptosis or cell death in colon cancer cells.