We previously identified Tbc1d23 as a candidate novel regulator of innate immunity using comparative genomics RNA interference screens in Caenorhabditis elegans and mouse macrophages. Using Tbc1d23 knockout mice and macrophages engineered to overexpress Tbc1d23, we now show that Tbc1d23 is a general inhibitor of innate immunity signaling, strongly inhibiting multiple TLR and dectin-signaling pathways. Tbc1d23 likely acts downstream of the TLR-signaling adaptors MyD88 and Trif and upstream of the transcription factor XBP1. Importantly, like XBP1, Tbc1d23 affects the maintenance, but not the initiation, of inflammatory cytokine production induced by LPS. Tbc1d23 acts as a RAB-GAP to regulate innate immunity signaling. Thus, Tbc1d23 exerts its inhibitory effect on innate immunity signaling in a spatiotemporal fashion. The identification of a novel spatiotemporal regulator of innate immunity signaling validates the comparative genomics approach for innate immunity gene discovery.