This study was designed to define some of the mechanisms by which rapamycin (RAPA), an mTOR inhibitor, induces hypertriglyceridemia when used as an immunosuppressive or antiproliferative agent and to determine whether low doses result in less undesirable side effects. Thirty male guinea pigs (n=10 per group) were randomly assigned to control (no RAPA), low-RAPA (0.08 mg/d), or high-RAPA (0.85 mg/d) treatment for 3 weeks. Rapamycin treatment resulted in more than a 2-fold increase in plasma triglycerides (TG) (P<.01), whereas no differences were observed in plasma cholesterol between RAPA and control groups. Low-RAPA treatment resulted in lower concentrations of cholesterol in the aorta (28.6%) and lower hepatic acyl-CoA cholesteryl acyltransferase activity compared to control and high-RAPA groups (P<.01). In addition, acyl-CoA cholesteryl acyltransferase activity was positively correlated with aortic cholesterol (r=0.43, P<.05). In contrast, aortic TG concentrations were higher in RAPA-treated guinea pigs than in control (P<.01). Very low density lipoprotein and low-density lipoprotein particles isolated from guinea pigs treated with RAPA were larger in size and contained more TG molecules than particles from control animals. Interestingly, plasma free fatty acids and fasting plasma glucose were 65% and 72% higher in the high-RAPA group than in control (P<.01). Tumor necrosis factor-alpha concentrations in the aorta were 3.6- and 10.4-fold higher in the low-RAPA and high-RAPA groups than in control guinea pigs (P<.01). These results suggest that RAPA interferes with TG metabolism by altering the insulin signaling pathway, inducing increased secretion of very low density lipoprotein and promoting deposition of TG in the aorta. Low RAPA was found to decrease cholesterol accumulation in tissue (liver and aorta) compared to high RAPA, suggesting that lower doses could be less detrimental to transplant patients.