Abstract Dysregulation of autophagy, an intracellular bulk degradation system, is strongly associated with the pathogenesis of a number of diseases, including cancer. Despite the identification of 31 genes that are essential for autophagy, the molecular machineries that regulate the generation and expansion of autophagosomal membranes are not fully understood. We have previously reported that Bif-1 interacts with Beclin 1 through UVRAG and that loss of Bif-1 suppresses the formation of autophagosomes during starvation. Here, we provide further evidence that Bif-1 acts with the PI3KC3 complex II (Beclin 1-UVRAG-PI3KC3; PI3KC3C2) to regulate the formation of autophagosomes by regulating the trafficking of the membrane-integrated, autophagy-related (Atg) protein, Atg9, by inducing the fission of Golgi membranes during the induction of autophagy. In response to starvation, Bif-1 co-accumulates with Atg9 in cytosolic foci that are positive for Golgi complex-derived membranes. Loss of Bif-1 or inhibition of the PI3KC3C2 not only suppresses the fragmentation of Golgi membranes and the Atg9-containing vesicles that are induced by nutrient starvation or rapamycin treatment, but also results in the accumulation of Atg9 at the Golgi complex. Importantly, Bif-1 mutants, which lack membrane binding and/or bending activity by deletion of the helix 0, helix 1 insertion, or both regions of the Bif-1 N-BAR domain, fail to restore rapamycin-induced formation of Atg9 foci and autophagosomes in Bif-1-deficient cells. Furthermore, immunoelectron microscopic analyses reveal that Atg9 localizes not only to autophagosomal membranes but also crescent-shaped isolation membrane-like structures during starvation. These results suggest that Bif-1, together with the PI3KC3C2, plays a key role in the fission of Golgi membranes, which generates Atg9-containing vesicles for the formation and expansion of autophagosomal membranes. 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 4835.