Studies conducted in mice have revealed positive effects of punicic acid (PUA). The aim of this study was to analyze the effects of PUA on fat accumulation and glycemic control in rats fed an obesogenic diet. Rats were randomly divided into two groups: control group and PUA group (diet supplemented with 0.5% PUA). No changes were observed in adipose tissue weights. The glucose tolerance test showed that the glycemic value in the PUA group had decreased significantly at the final time (120 min) (-19.3%), as had fructosamine levels (-11.1%). However, homeostasis model assessment (HOMA-IR) showed that insulin resistance did not improve. No changes were observed in the liver, skeletal muscle composition, or peroxisome proliferator-activated receptors (PPARs) activation. Low levels (mg/g tissue) of PUA (0.04 ± 0.01 in both tissues) and higher levels of cis-9,trans-11 conjugated linoleic acid (0.31 ± 0.08 in liver, 0.52 ± 0.11 in muscle) were found. PUA supplementation induced hypoplasia (-16.1%) due to the antiproliferative effect on hepatocytes. In conclusion, dietary supplementation of 0.5% PUA did not lead to decreased fat accumulation in adipose tissue, liver, or skeletal muscle, or to improved glycemic control. The hypoplasia induced in liver is a negative effect that should be considered before proposing PUA as a functional ingredient.