Macroautophagy or autophagy is an ubiquitous and conserved degradative pathway of cytosolic components, macromolecules or organelles, into the lysosome. By using biochemical and microscopic methods, which allow one to measure the rate of autophagy, the role of two regulators of Gi3 protein activity, activator of G-protein-signaling-3 (AGS3) and Galpha-interacting protein (GAIP), was studied in the control of autophagy in human colon cancer HT-29 cells. In HT-29 cells, autophagy is under the control of the Gi3 protein and, when bound to the GTP, the Galphai3 protein inhibits autophagy, whereas it stimulates autophagy when bound to the GDP. GAIP, which enhances the intrinsic GTPase-activating protein activity of the Galphai3 protein, stimulates autophagy by favoring the GDP-bound form of Galphai3. We showed that GAIP is phosphorylated on its serine 151 and that this phosphorylation is dependent on the presence of amino acids that modulate Raf-1 activity, the kinase upstream of Erk1/2. AGS3, a guanine nucleotide dissociation inhibitor, stimulates autophagy by binding Galphai3 proteins. The intracellular localization of AGS3 (Golgi apparatus and endoplasmic reticulum, two membranes known to be at the origin of autophagosomes) is consistent with its role in autophagy.