Here we report two additional families with hypoplastic amelogenesis imperfecta (AI) caused by pathogenic variants in ACP4, analyze the expression of Acp4 by mouse ameloblasts, and generate and characterize Acp4R110C knockin mice to better understand the pathogenesis of ACP4-associated enamel defects and deduce the role of ACP4 during tooth development. Here, ACP4 is linked to amelogenesis imperfecta.