Brown adipose tissue (BAT) is thermogenic, expressing high levels of uncoupling protein-1 to convert nutrient energy to heat energy, bypassing ATP synthesis. BAT is a promising therapeutic target for treatment of obesity and type 2 diabetes since it converts fatty acids into heat but mechanisms controlling brown adipogenesis remain unclear. Knockdown of acetyl-Coenzyme A acetyltransferase 1 (ACAT1) in C3H10T1/2 cells suppressed brown adipocyte maturation during the current study and ACAT1 overexpression promoted brown adipocyte maturation. The downstream target of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator-1-α (PGC1α), was involved in the action of ACAT1 on brown adipocyte maturation. ACAT1 overexpression enhanced AMPK phosphorylation and promoted PGC1α expression. It is suggested that ACAT1 promotes brown adipocyte maturation by activating the AMPK-PGC1α signaling pathway. •ACAT1 promoted differentiation of C3H10T1/2 cells into brown adipocytes.•ACAT1 activated the AMPK-PGC1α signaling pathway to enhance brown adipocyte maturation.•The mitochondrial location of ACAT1 was controlled by palmitoylation.