Background: Drosophila ubiquitin carboxy-terminal hydrolase L5 (Uch-L5) functions as a critical component of the 26S proteasome to mediate degradation of polyubiquitinated proteins. It was recently shown to modulate tissue/organ development by targeting the Smoothened protein in the hedgehog pathway. However, whether it plays a role in controlling organismal immune response remains largely unknown. Methods: Reverse transcription plus quantitative polymerase chain reaction (RT-qPCR), dual-luciferase, and Western blot assays were used to explore the potential function of Uch-L5 in the innate immune regulation in cultured Drosophila S2 cells. Further genetic manipulations and bacterial infections were conducted to confirm the findings in vivo. Results: Silencing of Uch-L5 antagonizes the immune deficiency (IMD) but not the Toll innate immune signaling both in vitro and in vivo. Moreover, Uch-L5 positively contributes to the Drosophila innate immune response via its N-terminal Uch domain, which is the catalytical triad executing its deubiquitinase activity. Conclusions: Our studies shed light on a novel function of the deubiquitinase Uch-L5 in governing the anti-microbial defense in Drosophila.