Abstract Viral vectors derived from different virus families, including poxvirus (canarypox virus vector ALVAC) and adenovirus (human Ad5 vector), have been widely used in vaccine development for a range of human diseases including HIV/AIDS. Less is known about the mechanisms underlying the host innate response to these vectors. Increasing evidence from clinical vaccine trials testing different viral vectors has suggested the importance of understanding basic elements of host–viral vector interactions. In this study, we investigated the innate interactions of APCs with two commonly used HIV vaccine vectors, ALVAC and Ad5, and identified AIM2 as an innate sensor for ALVAC, triggering strong inflammasome activation in both human and mouse APCs. Microarray and comprehensive gene-knockout analyses (CRISPR/Cas9) identified that ALVAC stimulated the cGAS/IFI16–STING–type I IFN pathway to prime AIM2, which was functionally required for ALVAC-induced inflammasome activation. We also provided evidence that, in contrast to ALVAC, the Ad5 vector itself was unable to induce inflammasome activation, which was related to its inability to stimulate the STING–type I IFN pathway and to provide inflammasome-priming signals. In preconditioned APCs, the Ad5 vector could stimulate inflammasome activation through an AIM2-independent mechanism. Therefore, our study identifies the AIM2 inflammasome and cGAS/IFI16–STING–type I IFN pathway as a novel mechanism for host innate immunity to the ALVAC vaccine vector.