Abstract: High stocking density in aquaculture leads to environmental degradation and affect animal health, ultimately caused by the imbalance of redox homeostasis. This study investigates the effects of reduced glutathione on the growth, antioxidant capacity and anti-heat stress ability of zebrafish (Danio rerio), as well as its mechanisms of action. Experimental diets containing 0, 0.2, 0.4, 0.6, and 0.8 g/kg reduced glutathione (rGSH), assigned as (control, 0.2 rGSH, 0.4 rGSH, 0.6 rGSH, and 0.8 rGSH), respectively, were fed to one-month-old D. rerio for six weeks. The results showed that supplementation of 0.6 rGSH promoted the growth, enhanced the glutathione peroxidase, glutathione reductase and upregulated the mRNA levels of tight junction proteins of the intestine of D. rerio, Significantly improvement of the survival rate of the fish grouped under 0.6 rGSH in 96 h acute high-temperature stress (P<0.05). Through a sterile D. rerio model, we found that rGSH not only directly acts on the D. rerio by increasing the expression level of nuclear translocated Nrf2 protein, and enhancing the antioxidant capacity and tight junction protein expression levels, but also enhance the physiological functions of D. rerio through modulating its gut microbiota. The 0.6 rGSH group had higher similarity in microbial composition and clustering compared to the control.It also significantly inceased the abundance of phylum Fusobacteriota and Cetobacterium genus in the 0.6 rGSH, whereas the abundance of Firmicutes and Bacteroidetes were reduced., Furthermore, for 0.6 rGSH group the biomarker bacterium was Cetobacterium a marker bacterium with significant enrichment of purine and pyrimidine metabolisms in KEGG pathways. Bugbase results show that the predominant phenotype for 0.6 rGSH was oxidative stress tolerance, mobile elements, anaerobic and facultative anaerobic types. Hence, this study clarified that 0.6 g/kg of rGSH will be applied to reduce the negative effects of heat stress of fish. This research offers a significant reference for the efficient utilization of reduced glutathione as a feed additive in aquaculture to enhance the overall health of aquatic animals by mitigating oxidative stress induced by adverse environmental factors in the aquaculture.