AbstractMicrobial products, such as lipopolysaccharide (LPS), can elicit efficient innate immune responses against invading pathogens. However, priming with LPS can induce a form of innate immune memory, termed innate immune “tolerance”, which blunts subsequent NF‐κB signaling. Although epigenetic and transcriptional reprogramming has been shown to play a role in innate immune memory, the involvement of post‐translational regulation remains unclear. Here, we report that ubiquitin‐specific protease 3 (USP3) participates in establishing “tolerance” innate immune memory through non‐transcriptional feedback. Upon NF‐κB signaling activation, USP3 is stabilized and exits the nucleus. The cytoplasmic USP3 specifically removes the K63‐linked polyubiquitin chains on MyD88, thus negatively regulating TLR/IL1β‐induced inflammatory signaling activation. Importantly, cytoplasmic translocation is a prerequisite step for USP3 to deubiquitinate MyD88. Additionally, LPS priming could induce cytoplasmic retention and faster and stronger cytoplasmic translocation of USP3, enabling it to quickly shut down NF‐κB signaling upon the second LPS challenge. This work identifies a previously unrecognized post‐translational feedback loop in the MyD88–USP3 axis, which is critical for inducing normal “tolerance” innate immune memory.