Spinal cord injury (SCI) causes long-term and severe disability, influencing the quality of life and triggering serious socioeconomic consequences. Lack of effective pharmacotherapies for SCI is largely attributable to an incomplete understanding of its pathogenesis. Caspase recruitment domain family member 6 (CARD6) was initially suggested to be a protein playing significant role in NF-κB activation. However, the effects of CARD6 on SCI progression remain unknown. In this study, the wild type (CARD6+/+), CARD6 knockout (CARD6-/-) and CARD6 transgenic (TG) mice were subjected to a SCI model in vivo, and in vitro experiments were conducted by treating microglia cells with lipopolysaccharide (LPS). Here, we identified CARD6 as a suppressor of SCI in mice. CARD6 knockout significantly accelerated functional deficits, neuron death and glia activation, whereas CARD6 overexpression resulted in the opposite effects. Both in vivo and in vitro SCI models suggested that CARD6 knockout markedly promoted apoptosis by increasing Cyto-c release to cytosol from mitochondria and activating Caspase-3 signaling. In addition, CARD6 knockout mice exhibited stronger inflammatory response after SCI, as evidenced by the significantly elevated expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6, which was largely through enhancing the activation of NF-κB signaling.