Testicular orphan nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily for which a ligand has not yet been found. In vitro data obtained from various cell lines suggest that TR4 functions as a master regulator to modulate many signaling pathways, yet the in vivo physiological roles of TR4 remain unclear. Here, we report the generation of mice lacking TR4 by means of targeted gene disruption (TR4^–/–). The number of TR4^–/– pups generated by the mating of TR4^+/– mice is well under that predicted by the normal Mendelian ratio, and TR4^–/– mice demonstrate high rates of early postnatal mortality, as well as significant growth retardation. Additionally, TR4^–/– females show defects in reproduction and maternal behavior, with pups of TR4^–/– dams dying soon after birth with no indication of milk intake. These results provide in vivo evidence that TR4 plays important roles in growth, embryonic and early postnatal pup survival, female reproductive function, and maternal behavior.