Signal transducers and activators of transcription 3 (STAT3) is constitutively active in human pancreatic cancer cells and can promote cell growth and apoptosis resistance that contribute to tumorigenesis. We determined if sorafenib, a multikinase inhibitor, can induce apoptosis by targeting STAT3 signaling to enhance apoptosis induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Human pancreatic cancer cell lines (PANC-1 and BxPC-3) were preincubated with sorafenib (Nexavar) alone or followed by TRAIL. Apoptosis was determined by Annexin V labeling, caspase cleavage, and Bax/Bak activation. Protein expression was analyzed by immunoblotting. Knockdown of STAT3, Mcl-1, and Bim were achieved by lentiviral small hairpin RNA. Adenoviral dominant-negative or retroviral constitutively active (CA) STAT3 were also used. Sorafenib inhibited constitutive STAT3 phosphorylation (Tyr(705)) and suppressed Mcl-1 and Bcl-x(L) proteins in a dose- and time-dependent manner. CA-STAT3 overexpression was shown to attenuate caspase-3 cleavage and suppression of Mcl-1 by sorafenib. STAT3 knockdown or a DN STAT3 was shown to downregulate Mcl-1 and Bcl-x(L) and to sensitize cells to TRAIL-mediated apoptosis. Treatment with sorafenib enhanced TRAIL-induced Annexin V staining and release of mitochondrial cytochrome c and AIF. Because the BH3-only Bim protein is a potent inducer of mitochondrial apoptosis, Bim knockdown was shown to attenuate caspase-3, caspase-9 cleavage, and Bax/Bak activation by sorafenib plus TRAIL. The suppression of STAT3 by genetic means or using sorafenib was shown to downregulate Mcl-1 and Bcl-x(L) and to sensitize cells to TRAIL-mediated apoptosis. These data indicate that targeting STAT3 may enhance treatment efficacy against pancreatic cancer.