This study investigated whether long-chain n-3 PUFA (LC n-3 PUFA) given to pregnant rats fed a high-fat (HF) diet may prevent fetal programming in male offspring at adulthood. Six weeks before mating, and throughout gestation and lactation, female nulliparous Sprague–Dawley rats were given a chow (C) diet, HF (60·6 % fat from maize, rapeseed oils and lard) or HF in which one-third of fat was replaced by fish oil (HF n-3). At weaning, the three offspring groups were randomly separated in two groups fed C diet, or HF without LC n-3 PUFA, for 7 weeks until adulthood. Glucose tolerance and insulin sensitivity were assessed by an oral glucose tolerance test both at weaning and at adulthood. Insulin signalling was determined in liver, muscle and adipose tissue by quantification of the phosphorylation of Akt on Ser 473 at adulthood. At weaning, as at adulthood, offspring from HF-fed dams were obese and displayed glucose intolerance (GI) and insulin resistance (IR), but not those from HFn-3 fed dams. Following the post-weaning C diet, phosphorylation of Akt was strongly reduced in all tissues of offspring from HF dams, but to a lesser extent in liver and muscle of offspring from HFn-3 dams. However, it was abolished in all tissues of all offspring groups fed the HF post-weaning diet. Thus, LC n-3 PUFA introduced in a HF in dams partially prevented the transmission of GI and IR in adult offspring even though they were fed without LC n-3 PUFA from weaning.