Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for red blood cell production and exert important cytoprotective effects on select vascular, immune, and cancer cells. To discover novel EPO action modes, we profiled the transcriptome of primary erythroid progenitors. We report Serpina3g /Spi2A as a major new EPO/EPOR target for the survival of erythroid progenitors. In knockout mice, loss of Spi2A worsened anemia caused by hemolysis, radiation, or transplantation. EPO-induced erythropoiesis also was compromised. In particular, maturing erythroblasts required Spi2A for cytoprotection, with iron and reactive oxygen species as cytotoxic agents. Spi2A defects were ameliorated by cathepsin-B/L inhibition, and by genetic co-deletion of lysosomal cathepsin B. Pharmacological inhibition of cathepsin B/L enhanced EPO-induced red cell formation in normal mice. Overall, we define an unexpected EPO action mode via an EPOR–Spi2A serpin–cathepsin axis in maturing erythroblasts, with lysosomal cathepsins as novel therapeutic targets.