In this study, the effects of 95% ethanol extracts of Euchresta formosana radix (EFR) on the cell cycle and apoptosis in human hepatocellular carcinoma (HCC) Hep3B cells were investigated. The results indicated that EFR decreased DNA synthesis and viable Hep3B cell numbers in a concentration-dependent manner. EFR induced a p21- and p27-dependent cell cycle arrest in S-phase and apoptosis of the Hep3B cells. The induction of apoptosis by EFR treatment was also confirmed by DAPI staining. EFR inhibited cyclin-dependent kinase (CDK)-1 and -2 expression and decreased cyclin B1 and E levels, resulting in S-phase arrest. EFR induced reactive oxygen species (ROS) production followed by endoplasmic reticulum (ER) stress that was based on the increase of GADD153 and GRP78 which led to the release of Ca2+ in the Hep3B cells. The EFR-promoted apoptosis was associated with increasing activation of caspases 3, 7, and 9 and enhanced poly(ADP-ribose) polymerase cleavage and increased expression of p21(CIP1/WAF1), p27(KIP1), Bax and Bad. Furthermore, the levels of Bcl-xl decreased after EFR treatment. Alteration of these key anti- and pro-apoptotic proteins could contribute to the increase in p53-independent apoptosis that was observed in the Hep3B cells.