MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression. For example, miRNAs repress gene expression by recruiting the miRNA-induced silencing complex (miRISC), a ribonucleoprotein complex that contains miRNA-engaged Argonaute (Ago) and the scaffold protein GW182. Recently, ubiquitin-protein ligase E3 component N-recognin 5 (UBR5) has been identified as a component of miRISC. UBR5 directly interacts with GW182 proteins and participates in miRNA silencing by recruiting downstream effectors, such as the translation regulator DEAD-box helicase 6 (DDX6) and transducer of ERBB2,1/2,2 (Tob1/2), to the Ago-GW182 complex. However, the regulation of miRISC-associated UBR5 remains largely elusive. In the present study, we showed that UBR5 down-regulates the levels of TNF receptor-associated factor 3 (TRAF3), a key component of Toll-like receptor signaling, via the miRNA pathway. We further demonstrated that p90 ribosomal S6 kinase (p90RSK) is an upstream regulator of UBR5. p90RSK phosphorylates UBR5 at Thr(637), Ser(1227), and Ser(2483), and this phosphorylation is required for the translational repression of TRAF3 mRNA. Phosphorylated UBR5 co-localized with GW182 and Ago2 in cytoplasmic speckles, which implies that miRISC is affected by phospho-UBR5. Collectively, these results indicated that the p90RSK-UBR5 pathway stimulates miRNA-mediated translational repression of TRAF3. Our work has added another layer to the regulation of miRISC.