A study on the nonspecific immunity of Litopenaeus vannamei ever inhabiting freshwater and seawater was carried out at different molt stages by comparing their total hemocyte count(THC) and respiratory burst(RB) and activity of phenol oxidase(PO), nitric oxide synthase(NOS) and lysozyme(LY). Two-way ANOVA showed that salinity and molt stage independently affected THC and RB and the activity of PO, NOS and LY of juvenile L. vannamei significantly(P < 0.05). The THC and RB and the activity of NOS gradually increased from the post-molt stages(A and B) to the pre-molt stages(D0–D3), which were common in shrimps inhabiting freshwater and seawater. The activity of PO peaked at the inter-molt stage(C), and touched the lowest at the post-molt stage in freshwater and pre-molt stage in seawater. The activity of LY was stable over the molt cycle. The RB and the activity of PO, NOS and LY of juvenile L. vannamei were significantly lower in freshwater than in seawater; whereas THC was significantly higher in freshwater than in seawater(P < 0.05). It was concluded that the post-molt stage(especially stage A) was critical to shrimp culture, which should be intensively attended when L. vannamei was cultured in freshwater. A study on the nonspecific immunity of Litopenaeus vannamei ever inhabiting freshwater and seawater was carried out at different molt stages by comparing their total hemocyte count (THC) and respiratory burst (RB) and activity of phenol oxidase (PO), nitric oxide synthase (NOS ) and lysozyme (LY). Two-way ANOVA showed that salinity and molt stage independently affected THC and RB and the activity of PO, NOS and LY of juvenile L. vannamei significantly (P <0.05). The THC and RB and the activity of NOS gradually increased from the post-molt stages (A and B) to the pre-molt stages (D0-D3), which were common in shrimps inhabiting freshwater and seawater. The activity of PO peaked at the inter-molt stage ), and touched the lowest at the post-molt stage in freshwater and pre-molt stage in seawater. The activity of LY was stable over the molt cycle. The RB and the activity of PO, NOS and LY of juvenile L. vannamei were significantly lower in freshwater than in seawater; but THC was significant ly was higher than freshwater than in seawater (P <0.05). It was concluded that the post-molt stage (especially stage A) was critical to shrimp culture, which should be intensively attended when L. vannamei was cultured in freshwater.