Autophagy mediates the degradation of cytoplasmic contents in the lysosome and plays a significant role in innate and adaptive immune responses. Lipid second messengers are implicated in the regulation of autophagy but the nature of the lipids involved and their mechanisms of action have yet to be characterized. Here we demonstrate a novel signaling role for diacylglycerol (DAG) in antibacterial autophagy. DAG production was necessary for efficient autophagy of Salmonella and its localization to bacteria-containing phagosomes preceded autophagy. Previous studies have revealed a role for the ubiquitin binding adaptor molecules p62 and NDP52 in autophagy of S. Typhimurium. We observed bacteria-containing autophagosomes colocalizing individually with either DAG or ubiquitinated proteins, indicating that both signals can act independently to promote anti-bacterial autophagy. We determined that the actions of phospholipase D (PLD) and phosphatidic acid phosphatase (PAP) were required for DAG generation and autophagy. The DAG-responsive δ isoform of protein kinase C was required for anti-bacterial autophagy, as were its downstream targets JNK and NADPH oxidase. Pkc1, the single PKC isoform in yeast, was essential for starvation-induced autophagy in Saccharomyces cerevisiae. These findings reveal an important role for DAG-mediated PKC function in mammalian anti-bacterial autophagy, and suggest a conserved role for PKC in autophagy regulation in eukaryotes.