Liver X receptor α (LXRα) and sterol regulatory element binding protein-1 (SREBP-1) are key transcription factors regulating lipid synthesis in mammals, but the joint function of LXRα activation and SREBP-1 maturation in milk fatty acid synthesis of lactating goat remains unclear. The objective of the present study was to determine the regulating function of LXRα and SREBP-1 in fatty acid metabolism of goat mammary gland using RT-qPCR. Goat mammary epithelial cells (GMEC) were treated by T0901317, a synthetic agonist of LXRα, to explore the regulatory function of LXRα-SREBP-1 pathway in goat mammary gland. The results showed that there was no significant change in the mRNA level of LXRα following treatment with different concentrations of T0901317, but a dose-dependent increase were observed for levels of mRNA, protein and relative luciferase activity of SREBP-1. Immunofluorescence results showed a clear accumulation of mature SREBP-1 in cell nucleus by the addition of T0901317. Activation of LXRα-SREBP-1 pathway increased significantly the mRNA expression of genes related to de novo fatty acid synthesis, fatty acid desaturation, elongation, transportation, and TG synthesis including FASN, ACCα, SCD1, IHD1, ACSS2, Elovl6, FABP3, DGAT1, DGAT2, AGPAT6, LPIN1 and PLIN2 (P < 0.01). One micro Mole T0901317 treatment caused a 20% increase of total content of cellular TG compared with the control (P < 0.01). Furthermore, addition of T0901317 remarkably increased the proportion of C16:1 and C18:1, while decreased that of C16:0 and C18:0. In conclusion, the LXRα regulates the expression and proteolytic maturation of SREBP-1 in GMEC, which implied that the crosstalk between LXRα and SREBP-1 may play an important role in the transcriptional regulation of de novo fatty acid synthesis and TG synthesis in goat mammary gland.