AbstractObjectiveImpaired adipogenic differentiation exacerbates metabolic disease in obesity. This study reported that high‐fat diet (HFD)‐fed mice housed at thermoneutrality exhibited impaired adipogenic differentiation, attributed to increased expression of histone deacetylase 9 (HDAC9). However, the impact of HFD on adipogenic differentiation is reportedly variable, possibly reflecting divergent environmental conditions such as housing temperature.MethodsC57BL/6J (wild‐type [WT]) mice were housed at either thermoneutral (28–30°C) or ambient (20–22°C) temperature and fed HFD or chow diet (CD) for 12 weeks. For acute exposure experiments, WT or transient receptor potential cation channel subfamily M member 8 (TRPM8) knockout mice housed under thermoneutrality were acutely exposed to ambient temperature for 6 to 24 h.ResultsWT mice fed HFD and housed at thermoneutrality, compared with ambient temperature, gained more weight despite reduced food intake. They likewise exhibited increased inguinal adipose tissue HDAC9 expression and reduced adipogenic differentiation in vitro and in vivo compared with CD‐fed mice. Conversely, HFD‐fed mice housed at ambient temperature exhibited minimal change in adipose HDAC9 expression or adipogenic differentiation. Acute exposure of WT mice to ambient temperature reduced adipose HDAC9 expression independent of sympathetic β‐adrenergic signaling via a TRPM8‐dependent mechanism.ConclusionsAdipose HDAC9 expression is temperature sensitive, regulating adipogenic differentiation in HFD‐fed mice housed under thermoneutrality.