Abstract: This study was conducted to investigate the effects of dietary fish meal (FM) replacement with Tenebrio molitor meal (TMM) at different inclusion levels on body composition, muscle amino acid and fatty acid profiles, antioxidant capacity, and immune performance of Pacific white shrimp (Litopenaeus vannamei). Four isonitrogenous and isolipidic experimental diets were formulated by replacing FM with TMM at 0 (TMM0, control), 15% (TMM15), 30% (TMM30), and 45% (TMM45) on an equal protein basis. Juvenile shrimp with an initial body weight of (0.42±0.01) g were randomly distributed into four experimental groups and fed the respective diets for 8 weeks under controlled laboratory conditions. The results demonstrated that the total amino acid content in the experimental diets increased linearly with increasing substitution levels of TMM, while the ratio of essential amino acids to total amino acids (EAA/TAAs) remained statistically unchanged among all dietary treatments. Notably, dietary linoleic acid content was elevated by 16.00% in the TMM45 group compared to TMM0 group. Conversely, eicosatetraenoic acid (EPA) and docosahexaenoic acid (DHA) levels were significantly decreased by 11.79% and 20.99%, respectively (P<0.05). In terms of growth performance, shrimp fed the TMM30 diet exhibited significantly higher weight gain rate and specific growth rate, which were improved by 12.34% and 3.47% compared to the control group, respectively (P<0.05), indicating that moderate replacement of FM with TMM promotes growth without adverse effects. For antioxidant capacity, hepatopancreas malondialdehyde content in the TMM45 group was significantly decreased, indicating reduced lipid peroxidation and oxidative stress. Concurrently, superoxide dismutase (SOD) and catalase (CAT) activities, as well as sod gene expression, were significantly upregulated (P<0.05), demonstrating enhanced antioxidant defense. In terms of immune performance, phenoloxidase and lysozyme activities, along with their corresponding gene expression, were significantly elevated with increasing dietary TMM substitution levels. Furthermore, key genes involved in the Toll/IMD signaling pathway (dor, rel), the JAK-STAT signaling pathway (stat, jun, fos), and antimicrobial peptide genes (cru, alf) were significantly upregulated in treatment groups compared to the control group (P<0.05), suggesting that TMM could activate innate immune responses through multiple signaling pathways. Regarding nutritional quality, no significant differences were observed in crude protein content of shrimp muscle among all treatment groups. However, the total content of flavor amino acids in the TMM45 group was significantly reduced compared to the TMM0 group (P<0.05). Interestingly, although dietary EPA and DHA levels decreased with increasing TMM inclusion, the concentrations of EPA and DHA in shrimp muscle were significantly elevated by 18.59% and 19.82% in the TMM45 group (P<0.05), suggesting a potential compensatory mechanism or selective retention of these long-chain polyunsaturated fatty acids. Additionally, crude lipid content in muscle increased significantly by 15.38% in the TMM45 group (P<0.05). Based on the economic benefit analysis, compared with the control group, the TMM30 group reduced feed costs by 8.01% while increasing profit by 35.28%. In conclusion, under isonitrogenous and isolipidic conditions, the optimal dietary inclusion level of TMM as a FM replacement for L. vannamei is 30%, which can not only reduce feed cost but also significantly promote shrimp growth performance, maintain muscle protein quality and the nutritional value of n-3 long-chain polyunsaturated fatty acids, and enhance antioxidant function and immune capacity through the activation of Toll/IMD and JAK-STAT signaling pathways. These findings provide theoretical support and practical guidance for the application of TMM in shrimp feed formulations, contributing to the sustainable development of aquaculture through reduced reliance on FM.