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Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, has been implicated in cardiometabolic inflammation; however, the intracellular signaling mechanisms linking TMAO to macrophage polarization remain incompletely defined. This study aimed to investigate whether TMAO promotes macrophage M1 polarization through a Piezo1-Yes-associated protein (YAP) signaling axis. Gain- and loss-of-function approaches were performed in RAW264.7 cells and primary bone marrow-derived macrophages, including Piezo1 overexpression, small interfering RNA-mediated knockdown of Piezo1 or YAP, and pharmacological inhibition of YAP. Macrophage polarization markers were evaluated by immunofluorescence, flow cytometry, real-time quantitative polymerase chain reaction, and Western blot analysis. Intracellular Ca²⁺ levels were assessed using Fluo-4 acetoxymethyl ester fluorescence imaging. TMAO increased inducible nitric oxide synthase expression and reduced arginase-1 levels (P < .01), accompanied by elevated IL-1β and IL-6 and decreased IL-4 and IL-10 (P < .01). Piezo1 overexpression promoted M1 polarization (P < .01), whereas Piezo1 knockdown under TMAO exposure attenuated inflammatory cytokine induction (P < .001). TMAO enhanced intracellular Ca²⁺ influx (P < .001) and reduced inhibitory phosphorylation of YAP (Ser127) (P < .01). Inhibition or silencing of YAP mitigated TMAO-induced M1 marker expression and downstream kappa B/extracellular signal-regulated kinase activation (P < .05). Similar results were confirmed in primary bone marrow-derived macrophages. These findings suggest that TMAO promotes macrophage M1 polarization in association with Piezo1-dependent Ca²⁺ influx and YAP activation, identifying the Piezo1-YAP axis as a potential mechanistic interface linking microbiota-derived metabolites to inflammatory remodeling. SIGNIFICANCE STATEMENT: This study identifies the Piezo1-Yes-associated protein signaling axis as a potential drug target in trimethylamine N-oxide-induced macrophage polarization toward a proinflammatory phenotype. Inhibition of this pathway attenuates inflammatory responses, suggesting a potential pharmacological strategy for microbiota-associated cardiovascular diseases.