This study tested the hypothesis that the effect of trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA) on energy intake (EI) and body weight (BW)/composition is confounded by dietary fat concentration and involves hypothalamic appetite-controlling mechanisms. ICR mice received low-fat (LF; 5 g/100 g) or high-fat (HF; 30 g/100 g) diets, with or without 0.5 g/100 g t10, c12 CLA (>98% pure) for 27 d. By d 13, BW and cumulative EI of the mice fed CLA supplemented LF diet (LF/CLA) were 6.6 and 23.6% lower, respectively, than the LF mice. In the subsequent 14 d, their EI rebounded and did not differ from the LF group. BW and EI did not differ between the HF and CLA supplemented HF (HF/CLA) groups. Hypothalamic pro-opiomelanocortin (POMC) mRNA expression was elevated (P = 0.031) on d 13 but suppressed (P < 0.001) on d 27 due to CLA treatment. CLA also suppressed AMP-activated protein kinase alpha2 expression. Mice in Expt. 2 received the LF diet, the LF/CLA, or were pair-fed the LF diet to the EI of the CLA group (LF/PF). LF/CLA and LF/PF mice did not differ in the hypothalamic POMC:neuropeptide Y expression ratio on d 13, but it was significantly lower in the LF/PF group on d 27. We conclude that the habitual dietary fat concentration influences the magnitude of weight loss induced by dietary t10, c12 CLA. The effect is in part independent of EI. Hypothalamic neuropeptides and nutrient sensing mechanisms may play a role.