Abstract TLR signaling is associated with the transcription of various proinflammatory cytokines, including TNF-α, IL-6, and IL-1β. After transcription, the mRNA of these proinflammatory cytokines needs to be tightly controlled at the posttranscriptional level to achieve an optimal expression. However, the precise mechanism of posttranscriptional regulation is not fully understood. In the current study, we found the expression of heterogeneous nuclear ribonucleoprotein U (hnRNP U), also termed scaffold attachment factor A, was greatly induced by TLR stimulation in macrophages. Knockdown of hnRNP U expression greatly attenuated TLR-induced expression of TNF-α, IL-6, and IL-1β, but not IL-12, whereas hnRNP U overexpression greatly increased TLR-induced expression of TNF-α, IL-6, and IL-1β. Furthermore, hnRNP U knockdown accelerated the turnover and decreased the t1/2 of TNF-α, IL-6, and IL-1β mRNA. RNA immunoprecipitation demonstrated that hnRNP U bound to the mRNA of these proinflammatory cytokines through the RGG motif. Importantly, we showed that TLR stimulation provided a stimulus for hnRNP U nuclear to cytoplasmic translocation. Therefore, we propose that hnRNP U induced by TLR signaling binds to the mRNA of a subset of proinflammatory cytokines and positively regulates the expression of these cytokines by stabilizing mRNA.