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Intracellular bacterial and viral diseases are widespread in the aquaculture industry and cause serious economic losses. Development of effective vaccines and adjuvants that can induce cell-mediated immunity is urgently needed for prevention of these diseases. Here we report the immunostimulatory effects of probiotic bacteria “E. faecalis” in ginbuna crucian carp Carassius auratus langsdorfii. Intraperitoneal injection of heat-killed E. faecalis induced an increase in CD4-1+ lymphocytes, CD8α+ lymphocytes and macrophages in vivo. Expression of Th1 cytokine genes was enhanced by exposure to the bacteria in vitro. We identified the leukocyte subsets that expressed specific Th1 cytokine genes: granulocytes and macrophages produced IL12 and IFNγrel2, respectively, while lymphocytes produced IFNγs including IFNγ1 and IFNγ2. Finally, expression of Th1 cytokines was also enhanced by intraperitoneal injection of heat-killed E. faecalis in vivo, while expression of Th2 cytokine was unchanged. Together, these findings suggest that heat-killed E. faecalis can induce cell-mediated immunity in fish.Heat-killed Enterococcus faecalis induced an increase in CD4-1+ lymphocytes, CD8α+ lymphocytes and macrophages in vivo.Heat-killed E. faecalis enhanced expression of Th1 cytokine genes including IFNγs, IFNγrels and IL12 in vitro.Stimulated granulocytes and lymphocytes by heat-killed E. faecalis in vitro expressed IL12 and IFNγs mRNA, respectively.Enhanced expression of Th1 cytokines by heat-killed E. faecalis was also observed in in vivo analysis.