The scope of this study was to assess the impact of calcium and conjugated linoleic acid (CLA) supplementation on plasma fatty acid profiles and to evaluate potential synergistic effects of both compounds against dietary obesity. Mice separated into five experimental groups were followed: control (C), high-fat diet (HF), HF with calcium (Ca), HF plus CLA and HF with both Ca and CLA. Plasma metabolites and fatty acids were determined by commercial kits and gas chromatography, respectively. Both dietary calcium and CLA supplementation contributed to lower body fat gain under a HF diet. Maximum efficacy was seen with calcium; no additional effect was associated with the combined treatment with CLA. Plasma leptin, adiponectin and HOMA index were in accordance with an altered glucose/insulin homeostasis in the HF and HF + CLA groups, whereas control levels were attained under Ca-enriched diets. Plasma fatty acids showed minor changes associated to CLA treatment, but a high impact on PUFA was observed under Ca-enriched diets. Our results show that the mechanism underlying the anti-obesity effects of calcium supplementation is mediated mainly by changes in PUFA plasma profile. In addition, the lack of synergy on body weight reduction in combination with associated lipid profiles of calcium and CLA suggests that calcium may interfere with absorption and/or bioactivity of CLA, which can be of relevance when using CLA-fortified dairy products against human obesity.