To date, more than twenty viruses require host-catalyzed de novo fatty acid biosynthesis for replication. In mammals, this pathway is driven by the enzyme fatty acid synthase (FASN), which condenses acetyl-CoA and malonyl-CoA to catalyze the production of the fatty acid palmitate. Palmitate serves as a precursor for various metabolic fates during viral infection, including lipid droplet formation for viral assembly, beta-oxidation for ATP generation, and/or post-translational modification of viral proteins. It was unknown if Mayaro virus (MAYV), an emerging mosquito-borne alphavirus that causes debilitating arthritogenic disease, requires FASN for replication. We found that pharmacological FASN inhibition with Fasnall, TVB-2640, and C75 significantly reduced MAYV infection in vitro. To determine the metabolic fate of palmitate, we pharmacologically inhibited the three pathways downstream of FASN and found that 2-bromopalmitate (2-BP), a protein palmitoylation inhibitor, led to a ~90% reduction in MAYV infection. Protein palmitoylation, or S-palmitoylation, is a post-translational modification in which palmitate is attached to sulfur atoms in cysteines residues. In Chikungunya virus , a related alphavirus, FASN-dependent palmitoylation of nonstructural protein 1 (nsP1) is essential for membrane association and formation of replication complexes. Based on this, we hypothesized that MAYV nsP1 is palmitoylated in a FASN-dependent manner. Protein labeling by click-chemistry confirmed that MAYV nsP1 is palmitoylated at conserved cysteine residues C417–C419. Using an alkyne acetate analog metabolized by FASN into alkyne palmitate, we observed specific labeling of wild-type nsP1 but not a cysteine-to-alanine mutant. Furthermore, treatment with FASN inhibitors or 2-BP abrogated alkyne acetate labeling of wild-type nsP1, reinforcing that MAYV protein palmitoylation is a FASN-dependent process. Our findings reveal a conserved mechanism of FASN-dependent protein palmitoylation in alphaviruses and highlight FASN as a potential therapeutic target for MAYV and related viruses.