Abstract: This study utilized zebrafish (Danio rerio) as a model to investigate the ameliorative effects and underlying mechanisms of dietary supplementation with the probiotic Lactobacillus rhamnosus on toxicity induced by tributyltin (TBT). Adult zebrafish were divided into three groups: a DMSO control group, a TBT group (2 μg/L TBT), and a TBT+PRO group (2 μg/L TBT supplemented with L. rhamnosus), and were fed accordingly for 21 days. The results showed that compared to TBT exposure alone, co-supplementation with L. rhamnosus significantly increased the abundances of Firmicutes and Lacticaseibacillus in the intestine; increased intestinal wall thickness and alleviated villi damage; reduced cerebral ventricle enlargement, and mitigated inflammatory cell infiltration and vacuolation in heart tissue. At the molecular level, the expression of pro-inflammatory factors (tnf-α, il-1β) was significantly downregulated, while the expression of the anti-inflammatory factor (il-10) was significantly upregulated in the intestine, brain, and heart. The expression of intestinal tight junction protein genes (claudin-2, zo-1) increased, leading to reduced intestinal permeability. Furthermore, lipopolysaccharide (LPS) levels were significantly decreased in the brain, serum, and intestine; the overexpression of hypothalamic-pituitary-interrenal (HPI) axis-related genes (crhr2, mr, pomc) was suppressed; the expression of key neuronal genes (α1-tubulin, bdnf, shha) was restored; cortisol (Cor) levels were significantly reduced; and the expression of apoptosis-related genes (caspase-3, caspase-9, bax) in the heart was significantly downregulated. These results indicate that L. rhamnosus can modulate the gut microbiota balance, reduce LPS levels, inhibit HPI axis overactivation, and alleviate inflammatory and apoptotic responses, thereby ameliorating TBT-induced damage to the nervous and cardiovascular systems in zebrafish. This study provides a theoretical basis and practical strategies for the bioremediation of TBT pollution and the application of probiotics in aquaculture.