Milk fat originates from the secretion of lipid droplets (LDs) synthesized within mammary epithelial cells. Adipocyte differentiation-related protein (ADRP; also known as PLIN2) is an LD binding protein that is crucial for synthesis of mature LD. The hypothesis was ADRP regulate LD production and metabolism in goat mammary epithelial cells (GMEC), thus, play a role in determining milk fat content. The objective of the present study was to investigate the function of ADRP in milk fat metabolism by ADRP overexpression and knockdown in GMEC using an adenovirus system. The expression of ADRP mRNA was determined by RT-PCR, and concentration of triacylglycerol (TG) extracted from GMEC was quantified on a micro-titer plate reader. Immunocytochemical staining revealed that ADRP localize to the surface of LDs. Supplementation with oleic acid (OA) enhanced its localization on the LDs surface and lipid accumulation. Overexpression of ADRP increased lipid accumulation and the concentration of triacylglycerol in GMEC. In contrast, morphological examination revealed that knockdown of ADRP decreased lipid accumulation even when OA was supplemented. This response was confirmed by the reduction in mass of cellular TG when ADRP was knockdown. The fact that knockdown of ADRP did not completely eliminate lipid accumulation at a morphological level in GMEC without OA suggested that some other compensatory factors may also aid in the process of LD formation. ADRP reversed the decrease of LD accumulation induced by Adipose triglyceride lipase (ATGL). This indicated that ADRP promote TG stability within LD by preventing access to ATGL. Collectively, these data provide direct in vitro evidence that ADRP play a key role in LD formation and stability in GMEC. Further experiments need to explore the mechanisms for enlargement of LD via ADRP activity in GMEC.