Humans differ in the outcome that follows exposure to life-threatening pathogens, yet the extent of population differences in immune responses and their genetic and evolutionary determinants remain undefined. Here, we characterized, using RNA sequencing, the transcriptional response of primary monocytes from Africans and Europeans to bacterial and viral stimuli—ligands activating Toll-like receptor pathways (TLR1/2, TLR4, and TLR7/8) and influenza virus—and mapped expression quantitative trait loci (eQTLs). We identify numerous cis-eQTLs that contribute to the marked differences in immune responses detected within and between populations and a strong trans-eQTL hotspot at TLR1 that decreases expression of pro-inflammatory genes in Europeans only. We find that immune-responsive regulatory variants are enriched in population-specific signals of natural selection and show that admixture with Neandertals introduced regulatory variants into European genomes, affecting preferentially responses to viral challenges. Together, our study uncovers evolutionarily important determinants of differences in host immune responsiveness between human populations. Display omitted •Human populations differ in their transcriptional responses to immune challenges•Cis- and trans-eQTLs contribute to population differences in immune responses•Immune-responsive regulatory variants have participated in human adaptation•Neandertals introduced variants affecting immune responses into European genomes Genetic variants enriched in population-specific signals of natural selection and, among Europeans, of Neandertal ancestry play a major role in the differences in transcriptional responses to inflammatory and infectious challenges observed between human populations.