A 10-week feeding trial was conducted to determine the requirements of vitamin D 3 in juvenile Litopenaeus vannamei at low salinity rearing conditions. The basal practical diet contained 685 IU vitamin D 3 per kg diet, Diets 2-6 contained 1960, 3240, 4220, 5630, and 7550 IU vitamin D 3 per kg diet, respectively. Diets were fed twice daily to triplicate groups of L. vannamei (initial average weight 0.39 ± 0.01 g). Growth performance and alkaline phosphatase activity in hepatosomatic were not affected by the supplementation of vitamin D 3 . Hepatosomatic index of shrimp fed diets containing vitamin D 3 at 685, 1960, and 3240 IU/kg was lower than the diet containing vitamin D 3 at 7550 IU. Whole-body moisture and zinc content were significantly decreased with increasing vitamin D 3 . Similarly, higher value was also observed on whole-body protein, ash content, calcium, and phosphorus and magnesium concentration with increasing vitamin D 3 . Results of the current study indicated that vitamin D 3 from ingredients could meet the growth requirement but not for whole-body mineral deposition. Broken-line regression analysis based on maximum whole-body ash indicated that vitamin D 3 requirement was recommended to be 6366 IU/kg for juvenile L. vannamei at low salinity rearing conditions.