Abstract: Background and Objective: Hypericum perforatum (HP) is known to have therapeutic effects in many types of cancer. Although the active substances hypericin and hyperforin in the composition of HP have been shown to exert apoptotic effects in cancer cell lines, research into the anticancer effects of the total extract of HP is meagre. The aim of this study was to investigate the proapoptotic effect of HP on human colorectal adenocarcinoma cell line HT29. Materials and Methods: HT29 cells (5×104 cells cm–2) were exposed to 99.2% medium and pharmacologically effective concentration of HP extract (0.8%) for 48 h. Cells were then washed in phosphate buffered saline PBS and lysed in RIPA buffer for 15 min on ice followed by centrifugation at 15000 rpm for 20 min and supernatants were taken and pellets were discarded. ELISA test was used to examine the expression and activity of cleaved caspase-3, Bax, Bcl-2, Wee1, GADD153, GRP78 and AIF proteins in HT29 cells. Results: ELISA test results indicated that the activities of proapoptotic proteins Bax, cleaved-caspase 3, Wee1, GRP78, GADD153 and AIF in human HT29 cell line were significantly increased in 48 h treatment with HP extract at 0.8% concentration. On the other hand, the activity of Bcl-2 was observed to decrease significantly. Conclusion: The results implicated that the HP extract in its original form effectively induces proapoptotic alterations in HT29 cells and a possible mutual signaling between the proapoptotic Wee1, GRP78, GADD153, AIF, Bax and cleaved caspase-3 proteins can contribute to this effect.