Abstract Background: Autophagy is a cellular response to adverse conditions that also plays a role in resistance to chemotherapy in cancer cells. Resistance renders systemic chemotherapy generally ineffective against human hepatocellular carcinoma (HCC). The HCC cell line Hep3B is deficient in key apoptotic regulators (p53, Fas) and shows differential apoptotic response to various chemotherapeutic agents. The antiglycolytic agent, 3-bromopyruvate (3-BrPA) is known to induce cell death in human HCC cell lines and has shown potent antitumor activity. In the current study we investigated the intracellular response of Hep3B cells to 3-BrPA treatment and compared it with another HCC cell line, SK-Hep1. Methods: We examined the intracellular effect of 3-BrPA by measuring endoplasmic reticulum (ER) stress using quantitative RT-PCR (qRT-PCR) and immunoblotting. The ultrastructural changes were observed by electron microscopy (EM). Apoptosis was evaluated by TUNEL assay and caspase-3 activation. Results: 3-BrPA treatment induced ER stress and inhibited protein synthesis in a dose and time dependent manner in both the HCC cell lines. EM revealed that 3-BrPA treatment resulted in the initiation of autophagy in Hep3B cells and apoptotic blebbing in SK-Hep1 cells. However, Hep3B cells overcame the initial autophagic response and finally progressed to apoptosis. Conclusions: SK-Hep1 cells upon 3-BrPA treatment underwent the classic pathway of apoptosis whereas Hep3B cells employed autophagy as a defense mechanism but failed to resist apoptosis. We conclude that 3-BrPA treatment promotes apoptosis in human HCC cell lines irrespective of their differential response associated with the status of key apoptotic regulators. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4462.