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The current study investigated a potential modulating effect of orally applied Lactobacillus rhamnosus 64 (LB64) during the early postnatal period (day of life: ∼3–30), during young adult period (day of life: 31–70) or throughout experiment, on parameters of trinitrobenzenesulfonic acid (TNBS)-induced colitis in adult rats. Treatment with LB64 during early postnatal, but not during young adult period reduced clinical damage score, neutrophil and macrophage infiltration into colon, the level of cytokine and myeloperoxidase (MPO) activity, but had no influence on other parameters of oxidative damage. Early postnatal treatment with LB64 also increased the diversity of fecal Bifidobacteria and Eubacteria, and improved maturation of ileal villi in 30-days old rats. When LB64 is applied during a critical period early in life, it affects immune system functioning of adults, probably by interactions with the mucosal immune system of the gastrointestinal tract that provides immune system maturation and shapes the overall immune response.