G-quadruplexes (G4s) regulate DNA replication and gene transcription, and are enriched in promoters without fully appreciated functional relevance. Here we show high selection pressure on putative G4 (pG4) forming sequences in promoters through investigating genetic and genomic data. Analyses of 76,156 whole-genome sequences reveal that G-tracts and connecting loops in promoter pG4s display lower or higher allele frequencies, respectively, than pG4-flanking regions, and central guanines (Gs) in G-tracts show higher selection pressure than other Gs. Additionally, pG4-promoters produce over 72.4% of transcripts, and promoter G4-containing genes are expressed at relatively high levels. Most genes repressed by TMPyP4, a G4-ligand, regulate epigenetic processes, and promoter G4s are enriched with gene activation histone marks, chromatin remodeler and transcription factor binding sites. Consistently,cis-expression quantitative trait loci (cis-eQTLs) are enriched in promoter pG4s and their G-tracts. Overall, our study demonstrates selective constraint of promoter G4s and reinforces their stimulative role in gene expression.