This study was conducted to investigate the effects of various levels of dietary mannan oligosaccharides (MOS) on growth performance, antioxidant capacity, immunity and intestinal microbiota of the Pacific white shrimp, Litopenaeus vannamei, at low salinity (salinity 3). Juvenile shrimp (0.68 ±0.01 g) were selected and fed experimental diets containing different MOS concentration (2, 4, or 8 g/kg) for 42 days. The shrimp fed 0 g/kg MOS diet at salinity 25 and 3 were served as seawater control and low salinity control, respectively. The results showed that: 1) The weight gain rate and specific growth rate of shrimp in low salinity control were significantly reduced compared with those in seawater control (P < 0.01). When compared with low salinity control, these parameters together with condition factor in 8 g/kg MOS group and specific growth rate in 4 g/kg MOS group were significantly increased (P < 0.05). 2) Compared with the seawater control, total superoxide dismutase activity and glutathione S-transferase activity of hepatopancreas in all low salinity groups were decreased significantly (P < 0.05). Malondialdehyde content in shrimp fed the 0 and 2 g/kg MOS diets were significantly increased (P < 0.05), while that returned to seawater control level in 4 and 8 g/kg MOS groups (P > 0.05). 3) α-diversity analysis showed that hyposaline stress had no significant effect on Shannon, Simpson, ACE and Chao1 indexes (P > 0.05), while ACE and Chao1 indexes in shrimp fed the 4 and 8 g/kg MOS diets were more significantly improved than two control groups (P < 0.05). In addition, β-diversity analysis showed that the intestinal microbiota of shrimp in seawter control was clearly separated from those 4 groups under hyposaline stress. 4) Compared with two control groups, the relative abundance of Proteobacteria in shrimp fed 2 and 4 g/kg MOS diets were significantly decreased (P < 0.05), while Firmicute and Verruccomicrobiota were increased significantly (P < 0.05). 5) Function prediction analysis by Tax4Fun showed that, compared with seawater control, low salinity stress had a subtle effect on intestinal microbial-mediated functions. While in 4 g/kg MOS group, most pathways related to energy metabolism and immunity at L3 level were significantly improved (P < 0.05). To sum up, all results suggest that dietary MOS can promote the growth performance, improve antioxidant capacity, and immune system in L. vannamei at low salinity, and shape intestinal microbiota structure and composition to help L. vannamei cope with hyposaline stress, and dietary 4 g/kg MOS supplementation can be optimal for the healthy culture of L. vannamei at low salinity.