Haploids and double haploids are important resources for studying recessive traits and have large impacts on crop breeding, but natural haploids are rare in animals.Mammalian haploids are restricted to gametes and are occasionally found in tumors with massive chromosome loss.Here we report the successful generation of haploid embryonic stem (ES) cells in mice and rats with both androgenetic and parthenogenetic origins.The androgenetic haploid ES (ahES) and parthenogenetic haploid ES (phES) cell lines maintain haploidy and stable growth over 30 passages,express pluripotent markers, possess the ability to differentiate into all three germ layers in vitro and in vivo, and can contribute to the germline of chimaeras when injected into blastocysts.Although epigenetically distinct from gametes, the ahES cells and phES cells can functionally replace sperms and oocytes, respectively, to produce viable and fertile animals.Such unique features of haploid embryonic stem cells make them serve as a convenient tool for gene functional studies and large-scale genetic Screening.Besides, we also show the application of gamete-like haploid stem cells to study the role of interaction of parental genomes in mouse early embryonic development.Our findings suggest that the haploid stem cells hold great application potentials in genetic and developmental studies.