Agouti signalling protein (ASIP) is a coat colour-related protein and also is a protein-related to lipid metabolism, which had first been found in agoutis. According to our previous study, ASIP is a candidate gene that affects the lipid metabolism in bovine adipocytes. However, its effect on milk lipid has not been reported yet. This study focused on the effect of the ASIP gene on the lipid metabolism of mammary epithelial cells in cattle. The ASIP gene was knocked out in bMECs by using CRISPR/Cas9 technology. The result of transcriptome sequencing showed that the differentially expressed genes associated with lipid metabolism were mainly enriched in the fatty acids metabolism pathways. Furthermore, the contents of intracellular triglycerides were significantly increased (p < 0.05), and cholesterol tended to rise (p > 0.05) in bMECs with the knockout of the ASIP gene. Fatty acid assays showed a significant alteration in medium and long-chain fatty acid content. Saturated and polyunsaturated fatty acids were significantly up-regulated (p < 0.05), and monounsaturated fatty acids were significantly decreased in the ASIP knockout bMECs (p < 0.05). The Q-PCR analysis showed that knockout of ASIP resulted in a significant reduction of gene expressions like PPARγ, FASN, SCD, and a significant up-regulation of genes like FABP4, ELOVL6, ACSL1, HACD4 prompted increased mid-to long-chain fatty acid synthesis. Overall, ASIP plays a pivotal role in regulating lipid metabolism in bMECs, which could further influence the component of lipid in milk.