Negative selection against deleterious alleles produced by mutation influences within-population variation as the most pervasive form of natural selection. However, it is not known whether deleterious alleles affect fitness independently, so that cumulative fitness loss depends exponentially on the number of deleterious alleles, or synergistically, so that each additional deleterious allele results in a larger decrease in relative fitness. Negative selection with synergistic epistasis should produce negative linkage disequilibrium between deleterious alleles and, therefore, an underdispersed distribution of the number of deleterious alleles in the genome. Indeed, we detected underdispersion of the number of rare loss-of-function alleles in eight independent data sets from human and fly populations. Thus, selection against rare protein-disrupting alleles is characterized by synergistic epistasis, which may explain how human and fly populations persist despite high genomic mutation rates.