Alanyl-tRNA synthetase 1 (AARS1) was recently identified as a lactyltransferase responsible for protein lactylation, a modification associated with epigenetic regulation and metabolic adaptation. AARS1 catalyzes lysine lactylation via an ATP- and lactate- dependent, but lactyl-CoA-independent mechanism. Here, we report compounds that inhibit AARS1 activity through an unconventional mechanism. Among them, XY353 covalently binds C184 of AARS1, inducing steric clashes with F175 and triggering structural rearrangements in the region that displace W176, a key residue for lactate binding. By means of a combination of ITC, SPR, MD, FP, enzymatic assays and structural analysis, we show that XY353 and its derivatives inhibit AARS1 by competing with lactate via the C184–F175–W176 relay, establishing a covalent allosteric mechanism of inhibition, which is further confirmed by our cellular data that XY353 derivatives XY353-1 reduces lactylation of the AARS1 substrate YAP and suppresses the proliferation of HGC-27 cells. Collectively, these findings define a covalent allosteric mechanism for AARS1 inhibition and provide the first chemical tools to explore its biological functions.