Honokiol (HNK) is an active component purified from Magnolia officinalis. HNK exhibits antitumor effects by inducing apoptosis and inhibiting the growth of many cancer cell lines, while proteins involved in antitumor effects in proteomic level are still unclear. In our study, HNK could inhibit HeLa cell proliferation and induce apoptosis in a concentration- and time-dependent manner. We utilized a quantitative proteomic technique termed SILAC (Stable isotope labeling with amino acids in cell culture)-MS (mass spectrometry) to study the differential proteomic profiling of HeLa cells treated by HNK. A total of 85 proteins were changed after HeLa cells were treated with 12 microg/ml HNK for 8 h, and 8 proteins showed up-regulation while 77 proteins down-regulated. The changed proteins were classified into 9 different categories, which covered a broad variety of cellular functions. In conclusion, HNK performs cytotoxicity to HeLa cells through co-operating of many proteins and different pathways.