Puberty/adolescence is a significant period of development and a time with a high emergence of psychiatric disorders. During this period, there is increased neuroplasticity and heightened vulnerability to stress and inflammation. The gut microbiome regulates stress and inflammatory responses and can alter brain chemistry and behaviour. However, the role of the gut microbiota during pubertal development remains largely uninvestigated. The current study examined gut manipulation with probiotics during puberty in CD1 mice on lipopolysaccharide (LPS)-induced immune responses and enduring effects on anxiety- and depression-like behaviours and stress-reactivity in adulthood. Probiotics reduced LPS-induced sickness behaviour at 12 h in females and at 48 h following LPS treatment in males. Probiotics also reduced LPS-induced changes in body weight at 48 h post-treatment in females. Probiotic treatment also prevented LPS-induced increases in pro- and anti-inflammatory peripheral cytokines at 8 h following LPS treatment, reduced central cytokine mRNA expression in the hypothalamus, hippocampus and PFC, and prevented LPS-induced changes to in the gut microbiota. A single exposure to LPS during puberty resulted in enduring depression-like behaviour in female mice, and anxiety-like behaviour in male mice in adulthood. However, pubertal exposure to probiotics prevented enduring LPS-induced depression-like behaviour in females and anxiety-like behaviors in males. Moreover, probiotics altered toll-like receptor-4 activity in the paraventricular nucleus of the hypothalamus (PVN) in males in response to a novel stressor in adulthood. Our results suggest that the gut microbiome plays an important role in pubertal neurodevelopment. These findings indicate that exposure to probiotics during puberty mitigates inflammation and decreases stress-induced vulnerabilities to emotional behaviours later in life, in a sex-specific manner.