MITA (also known as STING) is an ER-located adaptor protein, which mediates DNA-triggered innate immune response and is critically involved in autoimmune diseases and tumorigenesis. MITA is regulated by post-translational modifications, but how post-transcriptional mechanisms are involved in the regulation of MITA is still largely unknown. Here, we identified the RNA-binding protein LUC7L2 as a negative regulator of DNA virus-triggered innate immune response. LUC7L2-deficient mice exhibited resistance to lethal herpes simplex virus 1 (HSV-1) infection and reduced HSV-1 loads in the brain. Mechanistically, LUC7L2 directly bound to intron 3 of MITA precursor messenger RNA, inhibited its splicing and promoted its nonsense-mediated decay, leading to its downregulation at protein level. LUC7L2-deficient cells had markedly increased MITA level, leading to heightened innate antiviral response. Finally, LUC7L2 was induced following HSV-1 infection. Our findings reveal a feedback negative post-transcriptional regulatory mechanism for regulation of MITA-mediated innate immune response to viral and aberrant cellular DNA.