Stimulator of interferon genes (STING) is essential for innate immune pathway activation in response to pathogenic DNA. Proper activation of STING signaling requires STING translocation and phosphorylation. Here, we show that dual serine/threonine and tyrosine protein kinase (DSTYK) directly phosphorylates STING Ser366 at late endosomes to promote the activation of STING signaling. We find that TBK1 promotes STING post-Golgi trafficking via its kinase activity, thereby enabling the interaction between DSTYK and STING. We also demonstrate that DSTYK and TBK1 can both promote STING phosphorylation at late endosomes. Using an in vivo Dstyk-knockout model, we showed that mice deficient in DSTYK demonstrate reduced STING signaling activation and are more susceptible to infection with a DNA virus. Together, we reveal the previously unknown cellular function of DSTYK in phosphorylating STING and our findings provide insights into the mechanism of STING signaling activation at late endosomes.