Preterm birth is a major determinant of neonatal mortality and morbidity, affecting approximately one-third of preterm births as a result of prelabor rupturing of membranes. Infection and inflammation have strong causal links to preterm delivery, resulting in the activation of nuclear factor-kappaB (NFKB) and its downstream targets. Human sirtuin (SIRT) 6, which has ADP-ribosyl transferase and deacetylase activity, exhibits anti-inflammatory actions. The aims of this study were to determine the effect of 1) human preterm labor on SIRT6 expression in human gestational tissue and 2) the effect in primary amnion cells of SIRT6 inhibition, using small interfering RNA (siRNA) on prolabor mediators. To determine the effect of human preterm labor on SIRT6 expression, human fetal membranes were collected from women at preterm at the time of Cesarean section (no labor; n = 9) and from women after spontaneous labor and delivery (n = 9). SIRT6 mRNA and protein expression were significantly lower in fetal membranes after spontaneous preterm labor. Transfection of primary amnion cells with SIRT6 siRNA was associated with an increase in IL-1beta-induced proinflammatory cytokine gene expression and release (IL6, IL8, TNF [TNF-alpha]), cyclooxygenase ([COX]-2; official symbol PTGS2) expression and subsequent prostaglandin (PGE₂ and PGF_2alpha) release, and MMP9 gene expression and release of pro-MMP9. To determine whether SIRT6 affects NFKB transcriptional activity, primary amnion cells were transfected with NFKB tagged with luciferase and stimulated with IL1B. As expected, IL1B induced NFKB transcriptional activity. However, when cells were also cotransfected with a vector expressing SIRT6, there was a decrease in NFKB transcriptional activity. In conclusion, SIRT6 plays a role in regulating the terminal effector pathways of human labor and delivery via the NFKB pathway.