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In this study, Marsupeneaus japonicus microRNA-S5 (miR-S5) was found to be up-regulated 24 h post white spot syndrome virus (WSSV) or V. alginolyticus infection. The loss of function using an anti-microRNA oligonucleotide (AMO-miR-S5) showed that expression levels of multiple innate immune-related genes were affected. The expression of p53 and tumor necrosis factor-α (TNF-α) were significantly down-regulated, expression of myosin was significantly up-regulated. The miR-S5 knockdown delayed WSSV-induced death for 48 h, but the final mortality was not affected, while V. alginolyticus-induced mortality was increased by 30%. The effect of miR-S5 knockdown on phagocytosis and apoptosis rates showed that miR-S5 knock down significantly decreased phagocytosis rate of WSSV from 27.8% to 7.0%, and phagocytosis rate of V. alginolyticus from 27.2% to 21.4%, separately. WSSV-induced apoptosis decreased from 60.83% to 51.25%, but no effect on V. alginolyticus-induced apoptosis (43.72%–45.04%). We concluded that miR-S5 could be used by WSSV via regulating hemocyte phagocytosis and apoptosis processes, but helps to defend against bacterial infection by regulating the proPO system, superoxide dismutase activity and phagocytosis.Marsupeneaus japonicus microRNA-S5 was up-regulated 24 h after WSSV or V. alginolyticus infection.MiR-S5 could be used by WSSV via regulating hemocyte phagocytosis and apoptosis processes.MiR-S5 helps to defend against bacterial infection by regulating the proPO system, S activity and phagocytosis.