The aim of the present study was to determine whether culture supernatant and water soluble polysaccharides of L. rhamnosus PN04 could suppress the inhibition of human cancer cells along with the antioxidant activity. A culture supernatant and 90% ethanol extract (WSPES) of L. rhamnosus PN04 were studied on the antioxidant activity by using DPPH radical scavenging assay and using SRB assay for anticancer activity on two HeLa and Hep G2 human cancer cell lines. The amount of 90% ethanol extract change as the same the growth stage of L. rhamnosus PN04. The highest WSPES amount was obtained at the stationary phase. Both the culture supernatant (101.5×10⁶ cfu/ml) and 10 mg/ml WSPES (at stationary phase) were the most active antioxidant with the percent inhibition of  95.19 ± 0.062 and 52.86 ± 0.133 when compared with ascorbic acid at 29.77 µg/ml and 13.15 µg/ml before respectively. So, only culture supernatant and WSPES at this phase were performed the antitumor activity test. Culture supernatant of L. rhamnosus PN04 were determined to be less toxic to HeLa cell lines than were Hep G2 cell lines. However, the WSPES were more effective to Hep G2 than HeLa cell lines. Therefore, both culture supernatant and WSPES of this bacteria exhibited potent antioxidative activity and anticancer activity. The present study may contribute to discover the new and natural compounds for cancer treatment.