Background <p>Alpha-kinase 1 (ALPK1), a cytosolic receptor involved in innate immune activation, promotes apical trafficking in epithelial cells. While its role in autoinflammatory disorders is known, its function in epithelial homeostasis remains unexplored. This study investigates ALPK1’s role in murine lens development and its pathological relevance to congenital cataracts (CCs).</p> Methods <p>We utilized ALPK1-deficient (ALPK1^−/−) C57BL/6 mice and primary lens epithelial cells (LECs) with ALPK1 knockout (via lentiviral sgRNA) to analyze lens histo-morphological alterations and cellular and molecular pathologies, including apical protein transport and localization, endoplasmic reticulum (ER) stress, apoptosis, and LEC differentiation.</p> Results <p>We observed strong ALPK1 immunoreactivity in the LECs of C57BL/6 mice. ALPK1^−/− mice developed CCs with combined Y-suture and cortical opacities, disrupted lens cell architecture, and vacuolar degeneration. Molecular dysregulation included reduced phosphorylation of myosin Ia, mislocalization of zonula occludens-1 (ZO-1) from apical tight junctions to cytoplasmic aggregates, upregulation of the endoplasmic reticulum (ER) stress marker C/EBP-homologous protein (CHOP), increased apoptosis (evidenced by TUNEL-positive cells), and disorganized interlocking patterns in lens fiber cells. Lentiviral ALPK1 re-expression in ALPK1^−/− mice significantly restored lens transparency and ZO-1 apical localization, reduced CHOP expression, and suppressed apoptosis.</p> Conclusions <p>This study first demonstrates that ALPK1 is critical for maintaining LEC homeostasis by regulating myosin Ia phosphorylation-dependent apical trafficking and tight junction integrity. ALPK1 deficiency disrupts these processes, leading to loss of apical polarity, ER stress-induced apoptosis, and ultimately CC formation.</p> Graphical abstract <p></p>