CD38 contains an ADP ribosylcyclase domain that mediates intracellular Ca²⁺ signaling by production of cyclic ADP-ribose (cADPR), but the mechanisms by which agonists activate this enzyme remain unclear. The present study tested a hypothesis that a special lipid rafts (LRs) form, ceramide-enriched lipid platform, contributes to CD38 activation to produce cADPR in response to M₁ receptor stimulation in bovine coronary arterial myocytes (CAMs). By confocal microscopic analysis, oxotremorine (OXO), an M₁ receptor agonist, was found to increase LRs clustering on the membrane with formation of a complex of CD38 and LRs components such as GM₁, acid sphingomyelinase (ASMase) and ceramide, a typical ceramide-enriched macrodomain. At 80 µM, OXO increased LRs clustering by 78.8%, which was abolished by LR disruptors, methyl--cyclodextrin (MCD) or filipin. Using fluorescence resonance energy transfer (FRET) technique, 15.5 ± 1.9% energy transfer rate (vs. 5.3±0.9 of control) between CD38 and LR component, ganglioside M₁ was detected, further confirming the proximity of both molecules. In presence of MCD or filipin, there were no FRET signals detected. In floated detergent-resistant membrane fractions, CD38 significantly increased in LR fractions of CAMs treated by OXO. Moreover, MCD or filipin attenuated OXO-induced production of cADPR via CD38. Functionally, OXO-induced intracellular Ca²⁺ release and coronary artery constriction via cADPR were also blocked by LR disruption or ASMase inhibition. These results provide the first evidence that the formation of ceramide-enriched lipid macrodomains is crucial for OXO-induced activation of CD38 to produce cADPR in CAMs and these lipid macrodomains mediate transmembrane signaling of M₁ receptor activation to produce second messenger-cADPR.