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Introduction: Adolescence is a period of heightened susceptibility to anxiety disorders. Probiotic supplementation had a positive impact on reducing anxiety. The maternal microbiome plays an important role in child health outcomes and in the establishment of the offspring microbiome. Few studies have investigated the impact of gestational probiotic supplementation on the offspring's anxiety. Methods: The present study examined the impact of prenatal Lactobacillus helveticus NS8 supplementation (LAC) on Sprague Dawley rat offspring's anxiety‐like behavior. The behaviors tested in the present study include the elevated plus maze (EPM), the open field test (OFT), and prepulse inhibition (PPI). Analyses of variance were utilized. Results: (a) The performance of LAC adolescent rats in the EPM was similar to that in the OFT, both of which reflect that LAC caused an antianxiety effect in adolescent offspring rats and the antianxiety effect without sex differences; (b) LAC did not change performance in PPI and did not change the sex and age differences in PPI; and c. LAC decreased the body mass of rat offspring. Conclusion: Lactobacillus helveticus NS8 supplementation during gestation might have a moderate antianxiety effect in both males and females (especially adolescents) and be helpful for avoiding excessive body mass. [ABSTRACT FROM AUTHOR]