The study was designed to detect anticancer activity and proliferative effect of the Hardaliye in HT-29 and CF-1 cell lines, respectively. Based on MTT results, two dilutions namely, five (H5) and ten-fold (H10) were selected for further analyses. Apoptosis and antioxidant pathway related gene expression levels were detected using qRT-PCR and tali apoptosis assay was used to screen percentage levels of death, apoptotic and viable cells after 24h and 48h of treatment.Results suggested that Hardaliye was effective on cancer cells and trigger apoptosis via increase of Bax (22.45 ± 3.1-fold for H5 and 6.49 ± 0.1-fold for H10) as well as SOD and CAT activity. However, Hardaliye also affected slightly healthy cells when diluted fivefold, though, did not affect when dilute tenfold. According to gene expression levels, an increase at antioxidant enzyme and apoptosis inhibitors was seen after 24 h. On the other hand, antioxidant enzymes apart from GpX of H5, decreased after 48h. Predictably, antioxidant substances of Hardaliye revealed an antioxidant activity over time.Overall, this was the first report considering anticancer activity of Hardaliye besides its efficacy on healthy cells. In this respect, these preliminary results can indicate potential of Hardaliye as a food support in preventing colon cancer.