Ketosis, a common metabolic disease during early lactation, is associated with high circulating levels of β-hydroxybutyrate (BHB). A portion of BHB that reaches the mammary gland is utilized as precursor for synthesis of fatty acids. Recent findings from nonruminant studies revealed that long chain fatty acyl-CoA ligase 4 (ACSL4) could play a role in the regulation of cellular fatty acid metabolism, but the mechanisms by which ACSL4 mediates cellular lipid metabolism in response to BHB remains unclear. To achieve the aims, we conducted in vivo or in vitro analyses using bovine mammary gland biopsies and the immortalized mammary epithelial cell line (MAC-T). The in vivo study (n = 6 cows group^-1) involved healthy cows (plasma BHB < 0.60 mmol L^-1) or ketotic cows (plasma BHB > 2.0 mmol L^-1) from which mammary gland tissue was biopsied. In vitro, MAC-T cells were challenged with 0, 0.3, 0.6, 1.2, or 2.4 mmol L^-1 BHB for 24 h to determine an optimal dose. Subsequently, MAC-T were incubated with 1.2 mmol L^-1 BHB for 0, 3, 6, 12, 24, or 48 h. Furthermore, MAC-T cells were treated with small interfering ACSL4 (siACSL4) for 24 h or ACSL4 overexpression plasmid (pcACSL4) for 36 h followed by a challenge with 1.2 mmol L^-1 BHB for 24 h. Results showed that increased mRNA and protein abundance of lipogenic genes were linked to both mammary gland and in vitro challenge with BHB. BHB increased fatty acid content by activating ACSL4 expression, whereas inhibition of ACSL4 reduced BHB-induced reactive oxygen species (ROS) overproduction, enhancement of mitochondrial membrane potential, increase in fatty acid content, and lipid droplet accumulation. Furthermore, we also elevated ACSL4 expression with an overexpression plasmid to clarify its molecular role in response to BHB challenge. ACSL4 overexpression enhances BHB-induced lipid droplet accumulation by increased fatty acid content. Overall, the information showed that ACSL4 is crucial for the process of producing fatty acids from exogenous BHB. Reduced ACSL4 decreased fatty acid content and lipid droplet accumulation, improved mitochondrial function, directed more fatty acids towards oxidation. Thus, ACSL4 plays an important role in determining the fate of intracellular fatty acids and BHB in BMECs.