l-lactate modifies proteins through lactylation, but how this process occurs is unclear. Here we identify the alanyl-tRNA synthetases AARS1 and AARS2 (AARS1/2) as intracellular l-lactate sensors required for l-lactate to stimulate the lysine lactylome in cells. AARS1/2 and the evolutionarily conserved Escherichia coli orthologue AlaRS bind to l-lactate with micromolar affinity and they directly catalyse l-lactate for ATP-dependent lactylation on the lysine acceptor end. In response to l-lactate, AARS2 associates with cyclic GMP–AMP synthase (cGAS) and mediates its lactylation and inactivation in cells and in mice. By establishing a genetic code expansion orthogonal system for lactyl-lysine incorporation, we demonstrate that the presence of a lactyl moiety at a specific cGAS amino-terminal site abolishes cGAS liquid-like phase separation and DNA sensing in vitro and in vivo. A lactyl mimetic knock-in inhibits cGAS, whereas a lactyl-resistant knock-in protects mice against innate immune evasion induced through high levels of l-lactate. MCT1 blockade inhibits cGAS lactylation in stressed mice and restores innate immune surveillance, which in turn antagonizes viral replication. Thus, AARS1/2 are conserved intracellular l-lactate sensors and have an essential role as lactyltransferases. Moreover, a chemical reaction process of lactylation targets and inactivates cGAS.