Upon cytosolic viral DNA stimulation, cGMP-AMP synthase (cGAS) catalyzes synthesis of 2'3'cGMP-AMP (cGAMP), which binds to the adaptor protein MITA (mediator of IRF3 activation, also called STING, stimulator of IFN genes) and induces innate antiviral response. How the activity of MITA/STING is regulated to avoid excessive innate immune response is not fully understood. Here we identified the tyrosine-protein phosphatase nonreceptor type (PTPN) 1 and 2 as MITA/STING-associated proteins. PTPN1 and PTPN2 are associated with MITA/STING following viral infection and dephosphorylate MITA/STING at Y245. Dephosphorylation of MITA/STING leads to its degradation via the ubiquitin-independent 20S proteasomal pathway, which is dependent on the intrinsically disordered region (IDR) of MITA/STING. Deficiencies of PTPN1 and PTPN2 enhance viral DNA-induced transcription of downstream antiviral genes and innate antiviral response. Our findings reveal that PTPN1/2-mediated dephosphorylation of MITA/STING and its degradation by the 20S proteasomal pathway is an important regulatory mechanism of innate immune response to DNA virus.