In the present study, we synthesized a diglyceride (CLA-DG) from glycerol and conjugated linoleic acid (CLA). CLA-DG contained about 66% CLA as fatty acids. The safety of CLA-DG was assessed in acute and subchronic studies and in genotoxicity assays. In a single-dose acute study of CLA-DG, male and female rats were administered 5000 mg/kg body weight. CLA-DG caused no adverse effects and body weight gain and food consumption were within the normal range, thus the $LD_{50}$ of CLA-DG was determined to be greater than 5000 mg/kg. In a 90-day subchronic study (500, 1000, and 2000 mg/kg b.w./day, oral via gavage) in male and female rats, no significant adverse effects in food consumption, body weight, mortality, clinical chemistry, hematology, gross pathology, and histopathology were observed. CLA-DG did not show any potential to induce gene mutations in reverse mutation tests using Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2uvrA strains. CLA-DG did not induce chromosomal aberrations in cultured Chinese hamster lung cells. The results from these studies support the safety of CLA-DG.