Brown adipocytes cause an energy consumption by heat production and are thought to be a target for the prevention of obesity and related metabolic disorders. Piezo1 is a Ca²⁺-permeable non-selective cation channel and activated by mechanical stimuli. While, Piezo1 has been reported to be involved in mechano-sensation in non-sensory tissues, the expression and role of Piezo1 in brown adipocytes have not been well clarified. In this study, we evaluated a brown adipocytes line from UCP1-mRFP1 transgenic mice and analyzed this cell. Application of Yoda-1, a Piezo1 agonistsuppressed brown adipocytes differentiation in a dose-dependent manner. This suppression was significantly recovered by co-application with a Piezo1 antagonist and a calcineurin inhibitor. In addition, knock-down of Piezo1 impaired Yoda-1-induced suppression of brown adipocyte differentiation and application of Yoda-1 enhanced the calcineurin activity. These results suggest that activation of Piezo1 might suppress the differentiation through calcineurin pathway in brown pre-adipocytes.