Lipid accumulation alters macrophage biology and contributes to lipid retention within the vessel wall. In this study, we investigated the role of adipophilin on triglyceride accumulation and lipid‐droplet formation in THP‐1‐derived macrophages (THP‐1 macrophages). In the presence of acetylated low‐density lipoprotein, macrophages infected with an adenovirus expressing human adipophilin showed a 31% increase in triglyceride content and a greater number of lipid droplets compared with control cells. Incubation of macrophages with very low‐density lipoprotein (VLDL) dramatically increased cellular triglyceride content similarly in control and adipophilin‐overexpressing cells. By itself, VLDL increased adipophilin expression, which explains the lack of effect of adipophilin overexpression on cellular triglyceride content in macrophages loaded with VLDL. The lipid‐droplet content of macrophages was increased by overexpression of adipophilin and/or loading with VLDL. In contrast, inhibition of adipophilin expression using siRNA prevented lipid‐droplet formation and significantly reduced intracellular triglyceride content. Using inhibitors of β‐oxidation and acyl‐coenzyme A synthetase, results were obtained which suggest that adipophilin elevates cellular lipids by inhibition of β‐oxidation and stimulation of long‐chain fatty acid incorporation into triglycerides. Adipophilin expression in THP‐1 macrophages altered the cellular content of different lipids and enhanced the size of lipid droplets, consistent with a role for adipophilin in human foam cell formation.