Aims: Children born to obese mothers are predisposed to develop asthma but the mechanisms are unclear. We have shown in FVB/NJ mice that offspring of obese dams are prone to develop hyperinsulinemia and airway nerve-mediated reflex hyperresponsiveness. To rule out potentially biased results from inbred mouse models, we validated our study using diversity outbred (DO) mice, which more closely approximate the heterozygous genome of humans. Methods: Six-week old DO female mice were fed either a regular diet (17% fat) or a high-fat diet (45% fat) for 8 weeks before breeding, and throughout pregnancy and lactation. After weaning, all offspring were fed a regular diet. Body weight, body fat, and fasting blood glucose were measured in pregnant mice and 16-week-old offspring. Airway responsiveness to aerosolized 5-hydroxytryptamine (10-300 mM) was measured in anesthetized, paralyzed, and ventilated 16-week-old offspring. Results: Dams on a high-fat diet had more body fat than dams on a regular diet, but they had normal glucose tolerance before and during pregnancy, suggesting they did not have gestational diabetes. Offspring of dams on a high-fat diet had significantly higher body weight, body fat, fasting blood glucose and increased bronchoconstriction induced by 5-hydroxytryptamine than offspring of dams on a regular diet. Increased bronchoconstriction was blocked by vagotomy or atropine, demonstrating it was mediated by the vagal reflex. Conclusions: Maternal obesity increases reflex bronchoconstriction in offspring independent of genetic background that may involve hyperinsulinemia-altered airway neural function (Nie et al., 2014: PMCID:PMC4148040).