<p>Terminal erythropoiesis, the final stage of red blood cell maturation, is orchestrated by erythropoietin (EPO) and the master transcription factor, Kruppel-like factor 1 (KLF1). Recent studies highlight the importance of Ca²⁺ signaling in erythroid maturation; however, the underlying mechanisms remain elusive. Here we identify Orai1 as a novel EPO-responsive Ca²⁺ channel in erythroid cells, serving as a dynamic regulatory toggle that modulates KLF1 transcription and facilitates distinct phases of erythroid maturation. During the early stages, EPO-activated Orai1 suppresses KLF1 transcription through Ca²⁺-dependent NFAT2 activation and promoter binding, pausing erythroid maturation. As maturation progresses, Orai1 expression decreases, transitioning KLF1 regulation to an EPO–STAT5 pathway, thereby maintaining KLF1 expression and promoting terminal erythropoiesis. Using HUDEP-2 cells, umbilical cord blood and human pluripotent stem cell-derived CD71⁺ erythroblasts, we observed a progressive downregulation of Orai1 and reduction in intracellular Ca²⁺ levels during terminal maturation. The functional inactivation of Orai1 via R91W mutants and CRISPR–Cas9 knockout enhanced KLF1 expression, leading to increased erythroid-specific gene expression, accelerated erythroid maturation, higher levels of globin production and improved enucleation efficiency. This study unveils the EPO–Orai1–Ca²⁺–NFAT2–KLF1 axis as a critical regulatory checkpoint in erythropoiesis and highlights Orai1 downregulation as a potential strategy to enhance clinical red blood cell production by promoting erythrocyte maturation.</p>