To comprehensively evaluate the effects of Lactobacillus rhamnosus on the culture of Litopenaeus vannamei， as well as to determine the optimal form and dosage of its supplementation， seven experimental diets were formulated for juvenile L. vannamei ［（1.60±0.15） g］. These included control group （group CK， basal diet）， three live bacteria groups （LB1， LB2， LB3， with the diet supplemented with 1×10⁶， 1×10⁷， 1×10⁸ CFU/g of L. rhamnosus， respectively）， and three heat-inactivated bacteria groups （DB1， DB2， DB3， with the diet supplemented with 1×10⁶， 1×10⁷， 1×10⁸ CFU/g of heat-inactivated L. rhamnosus， respectively）. A 28-day culture trial was conducted to investigate the effects of L. rhamnosus on the growth performance and physiological health of L. vannamei. The results revealed the following： （1） Compared to the CK group， the weight gain rates of the LB1， LB2， LB3， and DB2 groups increased by 5.35%， 14.40%， 9.78%， and 6.11%， respectively （P<0.05）. （2） Intestinal amylase and lipase activities were significantly enhanced in all treatment groups （P<0.05）. （3） The ratio of n-3 to n-6 polyunsaturated fatty acids （PUFAs） in the muscles of the LB2 and DB2 groups increased significantly by 5.41% and 11.49%， respectively， compared with the CK group （P<0.05）. Furthermore， the total PUFA content in the DB2 group demonstrated a significant increase compared with the CK group （P<0.05）. （4） All L. rhamnosus supplemented groups exhibited enhanced serum phenoloxidase and lysozyme activities relative to the control （P<0.05）. Notably， the heat-inactivated groups exhibited a significant increase in total nitric oxide synthase activity （P<0.05）. （5） Acid phosphatase activity in the hepatopancreas of shrimp in the LB2， DB2， and DB3 groups increased significantly by 31.51%， 28.44%， and 22.12%， respectively （P<0.05）， while alkaline phosphatase activity increased in all groups except DB1 （P<0.05）. （6） Histological analysis revealed improved hepatopancreatic health in treatment groups， characterized by organized cellular arrangements and distinctive stellate luminal structures in hepatic tubules. In conclusion， dietary supplementation with L. rhamnosus significantly enhances growth performance， muscle quality， immune responses， and digestive enzyme activity in L. vannamei， while also improving hepatopancreatic histomorphology. Optimal effects were achieved at a concentration of 1×10? CFU/g for both viable and heat-inactivated formulations.