Abstract: Gill is an important respiratory organ of prawns, the main target organ of nitrite toxicity effect, and also one of the main enrichment sites of microplastics. The gill tissue participates in the physiological processes of prawns such as osmoregulation, nitrogen excretion, immune function, etc., which is important for maintaining the prawns' health. In order to investigate the effects of single and combined stress of nitrite and microplastics on the physiological functions of gill tissues of Litopenaeus vannamei, we designed the control group, 20 mg·L⁻¹ nitrite stress group (NIT), 10 μg·L⁻¹ microplastic stress group (MP), 20 mg·L⁻¹ nitrite and 10 μg·L⁻¹ microplastic composite stress group (NM), and then measured the changes of immune and osmotic regulation in gills of shrimps on the 14^th day. The results show that: 1) The oxidative stress indicators such as the contents of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) changed at different degrees after the stress. 2) The detoxification metabolic indicators such as the relative expression levels of cytochrome P450 (CYP450), glutathione S-transferase (GST) and apoptosis factor (CASP-3) were disturbed at different degrees after the stress. 3) The relative gene expression levels of the osmoregulation indexes such as ion transporters (VATP, NHE7, NKA-α, NKA-β and CA) and channel proteins (TIP4, CCP, CLC and AQP) occurred at different degrees of disorder after the stress. 4) The apoptosis index sucha as the relative expression level of apoptosis factor (CASP-3) gene decreased significantly in the three groups (P<0.05). Thus, it is inferred that nitrite and microplastic stress can induce the changes of immunity, detoxification metabolism and osmotic regulation in the gills of L. vannamei, affecting its normal physiological functions.