Lysine-63–linked (K63-linked) polyubiquitination of TRAF3 coordinates the engagement of pattern-recognition receptors withrecruited adaptor proteins and downstream activator TBK1 in pathways that induce type I IFN. Whether autoubiquitinationor other E3 ligases mediate K63-linked TRAF3 polyubiquitination remains unclear. We demonstrated that mice deficient inthe E3 ligase gene Hectd3 remarkably increased host defense against infection by intracellular bacteria Francisella novicida,Mycobacterium, and Listeria by limiting bacterial dissemination. In the absence of HECTD3, type I IFN response was impairedduring bacterial infection both in vivo and in vitro. HECTD3 regulated type I IFN production by mediating K63-linkedpolyubiquitination of TRAF3 at residue K138. The catalytic domain of HECTD3 regulated TRAF3 K63 polyubiquitination, whichenabled TRAF3-TBK1 complex formation. Our study offers insights into mechanisms of TRAF3 modulation and providespotential therapeutic targets against infections by intracellular bacteria and inflammatory diseases.