MicroRNA-26 (miR-26a and miR-26b) plays a critical role in lipid metabolism, but its endogenous regulatory mechanism in fatty acid metabolism is not clear in goat mammary epithelial cells (GMECs). GMECs with the simultaneous knockout of miR-26a and miR-26b were obtained using the CRISPR/Cas9 system with four sgRNAs. In knockout GMECs, the contents of triglyceride, cholesterol, lipid droplets, and unsaturated fatty acid (UFA) were significantly reduced, and the expression of genes related to fatty acid metabolism was decreased, but the expression level of miR-26 target insulin-induced gene 1 (INSIG1) was significantly increased. Interestingly, the content of UFA in miR-26a and miR-26b simultaneous knockout GMECs was significantly lower than that in wild-type GMECs and miR-26a- and miR-26b-alone knockout cells. After decreasingINSIG1expression in knockout cells, the contents of triglycerides, cholesterol, lipid droplets, and UFAs were restored, respectively. Our studies demonstrate that the knockout of miR-26a/b suppressed fatty acid desaturation by upregulating the targetINSIG1. This provides reference methods and data for studying the functions of miRNA families and using miRNAs to regulate mammary fatty acid synthesis.