Aim of this study was to investigate the antioxidant activity, cytotoxicity and antiproliferative effects of ethyl acetate extract of Penicillium chrysogenum var. chrysogenum (hPc.var.c) on the Caco-2 cells. hPc.var.c isolated from Lake Tuz in Turkey. Ethyl acetate extract was prepared from isolate of hPc.var.c culture medium. The free radical scavenging activity and total phenolic content were evaluated via DPPH and Folin Ciocalteu assays. Then cytotoxicity and cell proliferations were investigated with WST-1 method and real-time cell analysis system (RTCA-DP) on colorectal carcinoma (Caco-2) cells, respectively. Apoptosis/necrosis ratio was evaluated by using Annexin V-PI staining technique by the help of flow cytometry. In this study, free radical scavenging effect and total phenolic contend of hPc.var.c extract was found 73 +/- 0.34 % and 53 +/- 0.1 mg/GAE, respectively. All of the extract concentrations were demonstrated important cytotoxic and antiproliferative effects on colon adenocarcinoma cells according to concentration in a time dependent manner. IC50 concentration was determined 102 mu g/mL at the end of 72h by RTCA-DP system. The 25 mu g/mL concentration of the extract exhibited the highest apoptosis rate (47.2 %). In conclusion, it was observed that ethyl acetate extract of hPc.var.c had significantly anticancerogenic effects on Caco-2 cells. The hPc.var.c extract including secondary metabolites may have potential preventive and therapeutic effects against colon adenocarcinoma cells.