ABSTRACTWhile the signaling cascades and transcription factors that activate gene expression in macrophages following pattern recognition receptor engagement are well known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Recent phosphoproteomics analyses revealed that members of the SR and hnRNP families of splicing regulatory proteins undergo dynamic post-translational modification in infected macrophages. To begin to test if these splicing factors play a privileged role in controlling the innate immune transcriptome, we analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We observed that thousands of genes were up or downregulated in SR/hnRNP knockdown cells and differentially expressed genes (DEGs) varied significantly depending on the SR/hnRNP examined. We discovered that a subset of critical innate immune genes (Nos2, Mx1, Il1a) rely heavily on SR/hnRNPs for proper induction and/or repression, while others (Tnf, Il1b) are generally unaffected by splicing factor knockdown. We also discovered that many key immune sensors and signaling molecules are subject to regulation by alternative splicing. While our data does not provide evidence for positive correlation between a transcripts’ reliance of SR/hnRNPs for proper expression and the gene’s induction level, length, or intron/exon architecture, we found that many rapidly induced primary response genes are repressed by SR/hnRNPs. Consistent with SR/hnRNP proteins contributing to innate immune outcomes, knockdown of hnRNP K and U significantly enhanced the ability of RAW 264.7 macrophages to control viral replication. Based on these collective findings, we conclude that many innate immune genes have evolved to rely on one or more splicing regulatory factors to ensure the proper timing and magnitude of their induction, supporting a model wherein pre-splicing is a critical regulatory node in the innate immune response.