Card9 is a signaling adaptor protein in the downstream of many innate pattern recognition receptors(PRRs) and exerts a significant role in antifungal immunity. To date, Card9 deficiency has been reported to be related to increased susceptibility to many fungal infections. In this study, we established mucormycosis murine model of Rhizopus arrhizus (R. arrhizus) using wild-type(WT) mice and Card9 knockout (Card9 ) mice to investigate the antifungal effect of Card9 against R. arrhizus infection. Card9 mice were more susceptible to R. arrhizus infection than WT mice, which could be related to the impaired NF-κB pathway activation, local cytokine production and Th cell responses in Card9 mice.