Motile cilia and flagella are evolutionarily conserved organelles, and their defects cause primary ciliary dyskinesia (PCD), a disorder characterized by systemic organ dysfunction. The nexin-dynein regulatory complex (N-DRC) is a crucial structural component of motile cilia and flagella, present across various species from Chlamydomonas to humans. Defects in N-DRC components lead to multiple PCD symptoms, including sinusitis and male infertility. However, the phenotypic expression of N-DRC defects varies significantly among individuals, and there has been a lack of systematic study of core N-DRC components in mammals. Utilizing Drc1-4 and Drc7 knockout mice, this study systematically reveals the roles and assembly process of core N-DRC components in ependymal cilia, respiratory cilia, and sperm flagella. The findings show that core N-DRC components are crucial for the survival of mice on a purebred genetic background. In mixed genetic background mice, N-DRC defects impair the motility of motile cilia and the stability of flagellar axonemes. Additionally, a novel role of the N-DRC specific component (A-kinase anchoring protein 3) AKAP3 in regulating sperm phosphorylation was discovered. Collectively, our results provide a comprehensive understanding of the core N-DRC components in mammalian cilia and flagella.