<p><i>Lactobacillus plantarum</i>, a well-characterized probiotic from terrestrial animals, shows promising effects on fish growth and lipid metabolism. However, its role in promoting protein deposition in fish remains unclear. In this study, 180 juvenile Nile tilapia were randomly assigned to two groups and fed for eight weeks with either a control diet (CK) or a diet supplemented with <i>L. plantarum</i> (LR). The results showed that <i>L. plantarum</i> significantly improved final body weight, weight gain rate, body length, carcass rate, intestinal index, whole-body protein content, and villus length (<i>P</i> &lt; 0.05), accompanied by increased expression of growth-related genes and activation of the mechanistic target of rapamycin (mTOR) pathway. Conversely, the hepatosomatic index, condition factor, mesenteric fat index, hepatic lipid droplets, liver and serum triglycerides were significantly reduced (<i>P</i> &lt; 0.05). The <i>L. plantarum</i> group exhibited downregulated expression of lipogenic genes and upregulation of genes involved in lipolysis, β-oxidation, and fatty acid transport (<i>P</i> &lt; 0.05). Gut microbiota analysis revealed higher α-diversity and increased relative abundances of <i>Pseudomonas</i> and <i>Diaphorobacter</i>, while <i>Cetobacterium</i>, <i>Aestuariivirga</i>, and <i>Aurantimicrobium</i> at the genus level were reduced (<i>P</i> &lt; 0.05). Metabolomic profiling showed elevated bile acids, uridine, phosphatidylcholine, and carnitine derivatives (<i>P</i> &lt; 0.05). To confirm strain-specific effects, germ-free zebrafish were mono-colonized with <i>L. plantarum</i>, leading to suppressed lipogenesis and enhanced lipolysis and growth-related gene expression. Overall, <i>L. plantarum</i> promoted growth while simultaneously reducing fat accumulation, indicating a non-adipogenic growth-promoting effects in Nile tilapia.</p> Graphical Abstract <p></p>