MicroRNAs (miRNAs) are studied as key regulators of gene expression involved in different diseases. Several single nucleotide polymorphisms (SNPs) in miRNA genes or target sites (miRNA-related SNPs) have been proved to be associated with human diseases by affecting the miRNA-mediated regulatory function. To systematically analyze miRNA-related SNPs and their effects, we performed a genome-wide scan for SNPs in human pre-miRNAs, miRNA flanking regions, target sites, and designed a pipeline to predict the effects of them on miRNA–target interaction. As a result, we identified 48 SNPs in human miRNA seed regions and thousands of SNPs in 3′ untranslated regions with the potential to either disturb or create miRNA–target interactions. Furthermore, we experimentally confirmed seven loss-of-function SNPs and one gain-of-function SNP by luciferase assay. This is the first case of experimental validation of an SNP in an miRNA creating a novel miRNA target binding. All useful data were complied into miRNASNP, a user-friendly free online database (http://www.bioguo.org/miRNASNP/). These data will be a useful resource for studying miRNA function, identifying disease-associated miRNAs, and further personalized medicine. Hum Mutat 33:254–263, 2012. © 2011 Wiley Periodicals, Inc.