This study investigated the dietary thiamine (vitamin B1 ) requirement of P. vannamei post- larvae and its effects on growth performance, feed utilization, innate immunity, digestive enzyme activity and ammonia stress tolerance. High-performance liquid chromatography of the test diets revealed that the thiamine concentration of T0, T20, T40, T60, T80 and T100 experimental diets at 6.14, 28.5, 48.1, 73.3, 85.7 and 106 mg/kg of diet, respectively. A total of 1200 post- larvae (5 mg) were randomly distributed into 24 acryl tanks (50 shrimp/tank, 10 L) with four replicates per treatment. Daily feed requirement was divided into six equal portions and the feeding was done at 08:00, 10:00, 12:00, 14:00, 16:00 and 18:00 h for 30 days. After the feeding trial, 60 shrimp from each dietary treatment were captured, pooled and redistributed into three replicate tanks (10 L) for the ammonia stress challenge test. Thiamine supplemented groups showed a significant improvement in growth performance and feed utilization. Final body weight of T40, T60, T80 and T100 dietary groups were significantly higher compared to the T0 group and both weight gain percentage (WG%) and specific growth rate followed a similar trend. Significant enhancements in feed conversion ratio were observed in T40, T60, T80 and T100 groups and protein efficiency ratio was significantly elevated in thiamine supplemented groups (T60, T80 and T100) compared to the control group (T0). Survival percentage was not significantly different among the experimental groups. Thiamine supplementation significantly enhanced the activity of digestive enzymes (amylase, lipase and pepsin) and the significantly lowest activities were observed in T0 group. Compared to the thiamine deficient group, relative gene expression of insulin-like growth factor (IGF)-1 and IGF-binding protein gene expressions were significantly upregulated in thiamine supplemented groups. Antioxidative gene expressions (catalase, superoxide dismutase and glutathione peroxidase) were significantly upregulated through thiamine supplementation. Similarly, innate immune-related genes (prophenoloxidase and crustin ) were significantly expressed in thiamine supplemented groups. In the challenge test, after 48 h, a significantly higher survival rate was observed in T80 dietary group while thiamine deficient group manifested the significantly lowest survival. A broken line regression of WG% estimated the dietary thiamine requirement of P. vannamei post-larvae at 72.9 mg/kg of diet.