The objectives of the present study are to explore the function of SP1 in regulating triglyceride accumulation, droplet formation and the expression of genes associated with fat metabolism in goat mammary epithelial cells (GMECs). Adenovirus was used for SP1 overexpression, siRNA and mithramycin A were used for SP1 silencing and inhibition respectively. GC-MS was used for fatty acid composition analysis. Three repeats was used for qPCR and fatty acid determination. Six repeats were used for triglyceride determination and oil red O staining. The results were expressed as mean ± SD. Data of qPCR were analyzed using the 2^−ΔΔCt method. Group data for multiple comparisons were analyzed by ANOVA followed by Tukey's test. Significance was established at a P < 0.05. The results showed that overexpression and interference of SP1, using adenovirus and siRNA respectively, significantly reduced the content of cellular triglyceride and suppressed the accumulation of lipid droplets, and altered the expression of genes related to lipid metabolism. These data indicated that PPARγ and LXRα might have affected the role of SP1 in regulating other genes related to fatty acid uptake (CD36 and LPL), de novo fatty acid synthesis (FASN and ACACA), fatty acid elongation and desaturation (EVOLV6, SCD and FADS2), fatty acid transport (ACSL1, ACSS2 and FABP3), triglyceride synthesis (AGPAT6, LPIN1, DGAT1 and DGAT2), lipid droplet formation and secretion (TIP47, ADFP and XDH), and triglyceride hydrolysis (ATGL and HSL). But expression of SREBP1 was not affected by manipulation of SP1 despite of the crucial role in regulating de novo fatty acid synthesis. To support the results on triglyceride accumulation, adenovirus and mithramycin A, an inhibitor of SP1, was used to treat GMECs. The result showed that, similar to the decrease of triglyceride accumulation, both inhibition and overexpression of SP1 increased the relative content of saturated fatty acids, including C16:0, C14:0 and decreased the content of C18:1 which is important for triglyceride synthesis in GMECs. In conclusion, the findings provide a novel insight of SP1 function in regulating lipid metabolism in GMECs, and also provide novel method for dairy goat breeding.