Mammary gland involution involves a process that includes one of the most dramatic examples of cell death in an adult mammalian organism. We have previously shown that signal transducer and activator of transcription 3 (Stat3) regulates a lysosomal pathway of cell death in the first 48 h of involution and induces lysosome leakiness in mammary epithelial cells. Interestingly, Stat3 is associated also with the striking induction of autophagy that occurs concomitantly with cell death, presumably as a transient survival mechanism. The phosphatidylinositol 3-kinase regulatory subunits p55α and p50α are dramatically and specifically upregulated at the transcriptional level by Stat3 at the onset of involution. We show here that ablation of either Stat3 or p55α/p50α in vivo affects autophagy during involution. We used two different cell culture models (normal mammary epithelial cells and mouse embryonic fibroblasts) to further investigate the role of p55α/p50α in autophagy regulation. Our results demonstrate a direct role for p55α/p50α as inhibitors of autophagy mediated by p85α. Thus, Stat3 and its downstream targets p55α/p50α are key regulators of the balance between autophagy and cell death in vivo.