Our previous studies revealed that duck Tembusu virus (DTMUV) NS2A inhibited IFNβ signaling pathway by competitively binding to STING with TBK1, leading to reducing the phosphorylation of TBK1. Herein, we found that the 114-143 aa region of NS2A is critical for its interaction with STING and suppression of STING-mediated IFNβ signaling. We further identified the amino acids at positions L129, N130, L139, R140 and F143 of NS2A critical for NS2A-STING interaction. Subsequently, single residue substitution in the NS2A protein was introduced into the DTMUV replicon and infectious clone. The replicons with NS2A L129A and L130A mutations significantly inhibited viral genome RNA replication. The rDTMUV NS2A L129A, L139A and R140A mutant viruses yielded significantly lower titer levels than WT in both BHK-21 and DEF cells, with much more obvious effect on the viral genome level, and infectious virions formed outside of infected cells. Especially, the rDTMUV L129A mutant showed a significantly lower mortality in both embryos and ducks than WT. All NS2A-mutants decreased the weight gain of infected ducklings and reduced the viral loads in the spleen relative to WT. However, no significant differences of viral loads were observed in the blood, thymus, or liver. Our findings extend our previous study on the immune evasion role of flavivirus NS2A protein. The targeted therapy of disabling the viral strategies developed for evading innate defense can be applied to the development of attenuated flaviviruses.