Nuclear factor-kappa B (NF-κB)-induced inflammation plays a central role in the terminal process of human labor and delivery. Our previous studies show that IL1B induces NF-κB signaling through extracellular signal-regulated kinase (ERK; official gene symbol MAPK1), whereas TNF induces NF-κB-driven transcription of pro-labor mediators via an MAPK1-independent mechanism. Raf kinase inhibitor protein (RKIP) negatively regulates inflammation by inhibiting NF-κB activation directly or indirectly by inhibiting MAPK1. The role of RKIP in the processes of human labor and delivery is not known. The present study was performed to investigate the expression of RKIP in laboring and non-laboring human myometrium and determine the effect of siRNA knockdown of RKIP (siRKIP) on pro-labor mediators in human myometrial primary cells. Term labor was associated with a decrease in RKIP expression. Furthermore, RKIP expression was decreased in myometrial cells treated with IL1B and TNF, two likely factors contributing to preterm birth. The effect of siRKIP in primary myometrial cells was a significant augmentation of IL1B- and TNF-induced CXCL1 and CXCL8 mRNA abundance and secretion; PTGS2 mRNA levels and prostaglandin PGF2α release and MMP9 mRNA abundance and pro-MMP9 secretion. There was no effect of siRKIP on MAPK1 activation. On the other hand, RKIP knockdown was associated with increased activation of NF-κB RELA in the presence of IL1B and TNF. In conclusion, in human primary myometrial cells, RKIP negatively regulates IL1B- and TNF-induced expression and or secretion of pro-inflammatory and pro-labor mediators by inhibiting NF-κB RELA activation.