Honokiol (HNK) is a small organic molecule purified from magnolia species and has demonstrated anticancer activities in a variety of cancer cell lines; however, its effect on oral squamous cell carcinoma (OSCC) cells is unknown. We investigated the antitumor activities of HNK on OSCC cells in vitro for the first time. The inhibitory effects of HNK on the growth and proliferation of OSCC cells were demonstrated via in vitro 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide (PI) assays, and the apoptotic cells were investigated by the observation of morphological changes and detection of DNA fragmentation via PI, TdT-mediated dUTP-biotin nick end labeling (TUNEL), and DNA ladder assays, as well as flow cytometry assay. The results showed that HNK inhibited the growth and proliferation of OSCC cells in vitro in a time and dose-dependent manner. The inhibitory effect was associated with the cell apoptosis induced by HNK, evidenced by the morphological features of apoptotic cells, TUNEL-positive cells and a degradation of chromosomal DNA into small internucleosomal fragments. The study also demonstrated here that the inhibition or apoptosis mediated by 15 μg·mL-1 or 20 μg·mL-1 of HNK were more stronger compared with those of 20 μg·mL-1 5-fluorouracil (5-Fu, the control) applied to OSCC cells, when the ratio of OSCC cell numbers were measured between the treatment of different concentrations of HNK to the 5-Fu treatment for 48 h. HNK is a promising compound that can be potentially used as a novel treatment agent for human OSCC. Honokiol (HNK) is a small organic molecule purified from magnolia species and has introduced anticancer activities in a variety of cancer cell lines; however, its effect on oral squamous cell carcinoma (OSCC) cells is unknown. We investigated the antitumor activities of HNK on OSCC cells in vitro for the first time. The inhibitory effects of HNK on the growth and proliferation of OSCC cells were demonstrated via in vitro 3- (4,5-dimethyl thiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT ) and propidium iodide (PI) assays, and the apoptotic cells were investigated by the observation of morphological changes and detection of DNA fragmentation via PI, TdT-mediated dUTP-biotin nick end labeling (TUNEL), and DNA ladder assays, as well as flow cytometry assay. The results showed that HNK inhibited the growth and proliferation of OSCC cells in vitro in a time and dose-dependent manner. The inhibitory effect was associated with the cell apoptosis induced by HNK, evidenced by the morphological features of apoptotic cells, TUNEL-positive cells and a degradation of chromosomal DNA into small internucleosomal fragments. The study also demonstrated that that the inhibition or apoptosis mediated by 15 μg · mL-1 or 20 μg · mL- 1 of HNK were more reinforced compared with those of 20 μg · mL-1 5-fluorouracil (5-Fu, the control) applied to OSCC cells, when the ratio of OSCC cell numbers were measured between the treatment of different concentrations of HNK to the 5-Fu treatment for 48 h. HNK is a promising compound that can be potentially used as a novel treatment agent for human OSCC.