E74-like factor 5 (ELF5) is an Ets transcription factor of epithelial development, while the function of ELF5 gene in goat milk fat synthesis remains to be elucidated. In goat mammary epithelial cells, we performed RNA sequencing and analyzed differentially expressed genes (DEGs) after ELF5 gene overexpression. ELF5 gene significantly up-regulated the synthesis of triglyceride, total cholesterol, free fatty acid, and lipid droplets. We obtained 929 DEGs after ELF5 gene overexpression in GMECs. Among the DEGs, SPP1, ELOVL1, PNPLA2, FOXO1, PTGS2, SEMA6A, ACSL5, and GPNMB genes that are related to lipid metabolism were identified. Enrichment analysis showed MAPK and FoxO signaling pathways were up-regulated by ELF5 gene overexpression in GMECs. These findings offer evidence that ELF5 gene could be a candidate gene for the regulation of milk lipid synthesis in goats, and provide molecular targets for the breeding of goats with high milk fat.