Early life is considered a critical period for determining long-term metabolic health. Postnatal over-nutrition may alter glucocorticoid (GC) metabolism and increase the risk of developing obesity and metabolic disorders in adulthood. Our aim was to assess the effects of the dose and timing of a fish oil diet on obesity and the expression of GC-activated enzyme 11β-hydroxysteroid dehydrogenase type 1 (HSD1) in postnatal overfed rats. Litter sizes were adjusted to three (small litter (SL)) or ten (normal litter) rats on postnatal day 3 to induce overfeeding or normal feeding. The SL rats were divided into three groups after weaning: high-dose fish oil (HFO), low-dose fish oil (LFO) and standard-diet groups. After 10 weeks, the HFO diet reduced body weight gain (16 %, P<0·05), improved glucose intolerance and decreased hyperlipaemia levels (P<0·05) in SL rats, but the LFO diet did not have any effect on the same rats. Moreover, we chose postnatal week 3 (W3), 6 (W6) and 8 (W8) as the intervention time points at which to begin the 10-week HFO diet, and found that the HFO diet improved glucose utilisation and lipid metabolism at all time points. However, body weight of SL rats was reversed to normal levels by the post-weaning intervention (461 (sem 9·1) v. 450 (sem 2·0)). 11β-HSD1 mRNA expression in the adipose tissue (49 (sem 7·5) v. 161 (sem 18·3), P<0·05) and hepatic tissue (11 (sem 0·9) v. 16 (sem 1·5), P<0·05) was decreased by the HFO diet at W3, but not at W6 or W8 (P>0·05). In conclusion, the post-weaning HFO diet could reverse adverse outcomes and decrease tissue GC activity in postnatal overfed rats.