Tumor protein D52 (TPD52) is amplified/ over-expressed in cancers of diverse cellular origins. Altered cellular metabolism (including lipogenesis) is a hallmark of cancer development, and protein-protein associations between TPD52 and known regulators of lipid storage, and differential TPD52 expression in obese versus non-obese adipose tissue, suggest that TPD52 may regulate cellular lipid metabolism. We found increased lipid droplet numbers in stably TPD52-expressing BALB/c 3T3 cell lines, compared with control and TPD52L1-expressing cell lines. TPD52-expressing 3T3 cells showed increased fatty acid storage in triglyceride (from both de novo synthesis and uptake), and formed greater numbers of lipid droplets upon oleic acid supplementation than control cells. TPD52 co-localised with Golgi but not ER markers, and also showed partial co-localisation with Adrp-coated lipid droplets, with a proportion of TPD52 being detected in the lipid droplet fraction. Direct interactions between ADRP and TPD52, but not TPD52L1, were demonstrated using the yeast two-hybrid system, with ADRP/TPD52 interactions confirmed using GST pull-down assays. Our findings uncover a novel, isoform-specific role for TPD52 in promoting intracellular lipid storage, which may be relevant to TPD52 overexpression in cancer.