NR1D1 (also known as Rev-erbα) is a nuclear receptor and transcription factor that negatively regulates expression of circadian clock proteins. NR1D1 plays a crucial role in maintaining cellular lipid homeostasis by reducing lipogenesis and enhancing lipid oxidation in the liver and skeletal muscle, respectively. We previously reported that NR1D1 suppressed breast cancer growth and lung metastasis, accompanied by increased immune cell infiltration and activation. Given that tumors require the absorption of external lipids and de novo lipogenesis for growth and cellular construction, we explored the role of NR1D1 in lipid metabolism in breast cancer. We analyzed the global gene expression pattern in mammary tumors from Nr1d1 KO;PyMT mice and their control littermates by RNA-sequencing. Results showed that fatty acid and lipid metabolism-related genes were upregulated in the Nr1d1 KO;PyMT mice compared to those of Nr1d1 WT;PyMT mice. We observed elevated mRNA levels of enzymes related to fatty acid uptake and β-oxidation in mammary tumors of Nr1d1 KO;PyMT mice. Additionally, the in vivo results were confirmed in the NR1D1 knockout stable cell lines, revealing increased expression of fatty acid metabolism and lipogenesis-related proteins in the absence of NR1D1. Our findings suggest that NR1D1 regulates fatty acid metabolism in breast cancer, potentially contributing to the suppression of breast cancer progression.