Innate immunity represents one of the main host responses to viral infection.^1,2,3 STING (Stimulator of interferon genes), a crucial immune adapter functioning in host cells, mediates cGAS (Cyclic GMP-AMP Synthase) sensing of exogenous and endogenous DNA fragments and generates innate immune responses.⁴ Whether STING activation was involved in infection and replication of enterovirus remains largely unknown. In the present study, we discovered that human enterovirus A71 (EV-A71) infection triggered STING activation in a cGAS dependent manner. EV-A71 infection caused mitochondrial damage and the discharge of mitochondrial DNA into the cytosol of infected cells. However, during EV-A71 infection, cGAS-STING activation was attenuated. EV-A71 proteins were screened and the viral protease 2A^pro had the greatest capacity to inhibit cGAS-STING activation. We identified TRAF3 as an important factor during STING activation and as a target of 2A^pro. Supplement of TRAF3 rescued cGAS-STING activation suppression by 2A^pro. TRAF3 supported STING activation mediated TBK1 phosphorylation. Moreover, we found that 2A^pro protease activity was essential for inhibiting STING activation. Furthermore, EV-D68 and CV-A16 infection also triggered STING activation. The viral protease 2A^pro from EV-D68 and CV-A16 also had the ability to inhibit STING activation. As STING activation prior to EV-A71 infection generated cellular resistance to EV-A71 replication, blocking EV-A71-mediated STING suppression represents a new anti-viral target.