Abstract Cation homeostasis, in relation to various immune-suppressive diseases, is a novel field of investigation. Recently, patients with a loss-of-function mutation in magnesium transporter 1 (MAGT1) were reported to present a dysregulated Mg2+ homeostasis in T lymphocytes. Using Magt1-knockout mice (Magt1−/y), we show that Mg2+ homeostasis was impaired in Magt1−/y B cells and Ca2+ influx was increased after BCR stimulation, whereas T and NK cell function was unaffected. Consequently, mutant B cells displayed an increased phosphorylation of BCR-related proteins differentially affecting protein kinase C activation. These in vitro findings translated into increased frequencies of CD19+ B cells and marginal zone B cells and decreased frequencies of plasma cells among CD45+ splenocytes in vivo. Altogether, our study demonstrates for the first time, to our knowledge, that abolished MAGT1 function causes imbalanced cation homeostasis and developmental responses in B cells. Therefore, this study might contribute to a further understanding of B cell–related pathologies.