The protein LC3 is indispensible for the cellular recycling process of autophagy and plays critical roles during cargo recruitment, autophagosome biogenesis, and completion. Here, we report that LC3 is phosphorylated at threonine 50 (Thr(50)) by the mammalian Sterile-20 kinases STK3 and STK4. Loss of phosphorylation at this site blocks autophagy by impairing fusion of autophagosomes with lysosomes, and compromises the ability of cells to clear intracellular bacteria, an established cargo for autophagy. Strikingly, mutation of LC3 mimicking constitutive phosphorylation at Thr(50) reverses the autophagy block in STK3/STK4-deficient cells and restores their capacity to clear bacteria. Loss of STK3/STK4 impairs autophagy in diverse species, indicating that these kinases are conserved autophagy regulators. We conclude that phosphorylation of LC3 by STK3/STK4 is an essential step in the autophagy process. Since several pathological conditions, including bacterial infections, display aberrant autophagy, we propose that pharmacological agents targeting this regulatory circuit hold therapeutic potential.