Key points Calcium homeostasis modulator 1 (CALHM1), a new voltage‐gated ATP‐ and Ca2+‐permeable channel, plays important physiological roles in taste perception and memory formation. Regulatory mechanisms of CALHM1 remain unexplored, although the biophysical disparity between CALHM1 gating in vivo and in vitro suggests that there are undiscovered regulatory mechanisms. Here we report that CALHM1 gating and association with lipid microdomains are post‐translationally regulated through the process of protein S‐palmitoylation, a reversible attachment of palmitate to cysteine residues. Our data also establish cysteine residues and enzymes responsible for CALHM1 palmitoylation. CALHM1 regulation by palmitoylation provides new mechanistic insights into fine‐tuning of CALHM1 gating in vivo and suggests a potential layer of regulation in taste and memory. Abstract Emerging roles of CALHM1, a recently discovered voltage‐gated ion channel, include purinergic neurotransmission of tastes in taste buds and memory formation in the brain, highlighting its physiological importance. However, the regulatory mechanisms of the CALHM1 channel remain entirely unexplored, hindering full understanding of its contribution in vivo. The different gating properties of CALHM1 in vivo and in vitro suggest undiscovered regulatory mechanisms. Here, in searching for post‐translational regulatory mechanisms, we discovered the regulation of CALHM1 gating and association with lipid microdomains via protein S‐palmitoylation, the only reversible lipid modification of proteins on cysteine residues. CALHM1 is palmitoylated at two intracellular cysteines located in the juxtamembrane regions of the third and fourth transmembrane domains. Enzymes that catalyse CALHM1 palmitoylation were identified by screening 23 members of the DHHC protein acyltransferase family. Epitope tagging of endogenous CALHM1 proteins in mice revealed that CALHM1 is basally palmitoylated in taste buds in vivo. Functionally, palmitoylation downregulates CALHM1 without effects on its synthesis, degradation and cell surface expression. Mutation of the palmitoylation sites has a profound impact on CALHM1 gating, shifting the conductance–voltage relationship to more negative voltages and accelerating the activation kinetics. The same mutation also reduces CALHM1 association with detergent‐resistant membranes. Our results comprehensively uncover a post‐translational regulation of the voltage‐dependent gating of CALHM1 by palmitoylation.