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The present study assessed the protective effects and underlying mechanisms of mulberry leaf polysaccharides (MLPs) against hydrogen peroxide (H₂O₂)-induced oxidative stress injury in the peripheral blood leukocytes (PBLs) of <i>Megalobrama amblycephala</i>. Five treatment groups were established in vitro: the NC group (PBLs incubated in an RPMI-1640 complete medium for 4 h), the HP group (PBLs incubated in an RPMI-1640 complete medium for 3 h, and then stimulated with 100 μM of H₂O₂ for 1 h), and the 50/100/200-MLP pre-treatment groups (PBLs were pre-treated with MLPs (50, 100, and 200 μg/mL) for 3 h, and then stimulated with 100 μM of H₂O₂ for 1 h). The results showed that MLP pre-treatment dose-dependently enhanced PBLs’ antioxidant capacities. The 200 μg/mL MLP pre-treatment effectively protected the antioxidant system of PBLs from H₂O₂-induced oxidative damage by reducing the malondialdehyde content and lactic dehydrogenase cytotoxicity, and increasing catalase and superoxide dismutase activities (<i>p</i> < 0.05). The over-production of reactive oxygen species, depletion of nicotinamide adenine dinucleotide phosphate, and collapse of the mitochondrial membrane potential were significantly inhibited in the 200-MLP pre-treatment group (<i>p</i> < 0.05). The expressions of endoplasmic reticulum stress-related genes (forkhead box O1α (<i>foxO1α</i>), binding immunoglobulin protein (<i>bip</i>), activating transcription factor 6 (<i>atf6</i>), and C/EBP-homologous protein (<i>chop</i>)), Ca²⁺ transport-related genes (voltage-dependent anion-selective channel 1 (<i>vdac1</i>), mitofusin 2 (<i>mfn2</i>), and mitochondrial Ca²⁺ uniporter (<i>mcu</i>)), and interleukin 6 (<i>il-6</i>) and bcl2-associated x (<i>bax</i>) were significantly lower in the 200-MLP pre-treatment group than in the HP group (<i>p</i> < 0.05), which rebounded to normal levels in the NC group (<i>p</i> > 0.05). These results indicated that MLP pre-treatment attenuated H₂O₂-induced PBL oxidative damage in the <i>M. amblycephala</i> by inhibiting endoplasmic reticulum stress and maintaining mitochondrial function. These findings also support the possibility that MLPs can be exploited as a natural dietary supplement for <i>M. amblycephala,</i> as they protect against oxidative damage.