Objective: Cytokines, molecules within the immune system that affect either a pro- or anti-inflammatory response, have previously been shown to influence birth outcomes. The maternal cytokine gene–environment interactions are thought to alter their expression, potentially influencing susceptibility to adverse birth outcomes. The aim of this study was to determine the association between the maternal interleukin-1β (IL-1β) haplotype and expression variation with oxides of nitrogen (NO_x) levels, and thereafter investigate the IL-1β haplotype-specific effects of NO_x exposure levels, IL-1β mRNA expression and other variables on gestational age. Material and methods: Using the prospective Mother and Child in the Environment (MACE) birth cohort in Durban, South Africa, 335 participants were genotyped for the IL-1β haplotype. Previous studies showed that three single nucleotide polymorphisms (SNPs), IL-1β-1464G/C, -511C/T and -31C/T, constitute the IL-1β functional haplotype. These SNPs were genotyped using a restriction fragment length polymorphism assay, while IL-1β mRNA expression was measured using a quantitative real-time polymerase chain reaction assay. Individual estimates of NO_x exposure were obtained by land use regression modelling. A multivariate linear regression analysis was employed to test for significant effects on gestational age. Results: IL-1β mRNA expression was found to possess a haplotype-dependent effect ( p = 0.0001) and its expression levels positively correlated with NO_x levels ( r = 0.34; p = 0.006). In the high haplotype model, a unit increase in NO_x exposure level was associated with a decrease in gestational age by 1 week ( p = 0.02). Furthermore, gestational age decreased by 0.9 weeks for every unit increase of IL-1β mRNA expression level ( p = 0.025). HIV-1 positivity was associated with a 0.2-week decrease in gestational age ( p = 0.035) in the intermediate haplotype model and a 0.4-week decrease in the high haplotype model ( p = 0.044). Conclusion: These data have implications for better understanding the effect of prenatal NO_x exposure on gestational age and demonstrate the role of the IL-1β haplotype in modulating the effects of NO_x exposure.