An 8 weeks feeding trial was carried out to investigate the effects of dietary cholesterol content on the immune related genes and resistances to low temperature and low dissolved oxygen stress in white shrimp Litopenaeus vannamei cultured in freshwater. Five isonitrogenous and isoenergetic experimental diets (C0, C1, C2, C3, C4) were prepared to feed shrimps with initial average mass of (0.14±0.03) g and the measured cholesterol content of experimental diets was 0.78, 1.57, 2.45, 3.43 and 4.18 mg/g diet, respectively. The immune related genes (Toll receptor, IMD, and lysozyme) expressions before and after Vibrio parahaemolyticus infection, cumulative mortalities of the shrimp experienced acute low temperature and low dissolved oxygen stresses were detected. The results indicated that dietary cholesterol significantly affected the changes in expression levels of Toll receptor, IMD and lysozyme mRNAs of the shrimp experienced Vibrio challenge. During the 48 h after infection, the shrimp in C2 treatment displayed the largest variations in Toll receptor and lysozyme mRNAs expressions while the shrimp in C1 treatment displayed the largest variation in IMD mRNA expression, followed by shrimp in C2. All the treatments had the peaks of expressions of Toll receptor and lysozyme mRNAs at 24 h post infection. The shrimp in C2, C3 and C4 treatments displayed the peaks of IMD mRNA expression at 24 h post infection, while 42 h post infection for C0 and C1 treatments. Under the acute process cooling stress, the shrimp in C0 and C4 treatments displayed 100% mortality at 15℃ while the shrimp in C1, C2 and C3 treatments displayed 100% mortality at 14℃. There was no significant difference in oxygen consumption and lethal dissolved oxygen concentration between the groups under acute low dissolved oxygen stress. It is therefore suggested that dietary cholesterol of 2.45 mg/g could improve the immune sensitivity of L. vannamei cultured in freshwater, and the shrimp fed diet containing 1.57-3.43 mg/g cholesterol displays better resistance to low temperature stress.