Inorganic sulfate is required for numerous functions in mammalian physiology, and its circulating levels are proposed to be maintained by the Na⁺-SO₄^2- cotransporter, (NaS_i-1). To determine the role of NaS_i-1 in sulfate homeostasis and the physiological consequences in its absence, we have generated a mouse lacking a functional NaS_i-1 gene, Nas1. Serum sulfate concentration was reduced by >75% in Nas1^-/- mice when compared with Nas1^+/+ mice. Nas1^-/- mice exhibit increased urinary sulfate excretion, reduced renal and intestinal Na⁺-SO₄^2- cotransport, and a general growth retardation. Nas1^-/- mouse body weight was reduced by >20% when compared with Nas1^+/+ and Nas1^+/- littermates at 2 weeks of age and remained so throughout adulthood. Nas1^-/- females had a lowered fertility, with a 60% reduction in litter size. Spontaneous clonic seizures were observed in Nas1^-/- mice from 8 months of age. These data demonstrate NaS_i-1 is essential for maintaining sulfate homeostasis, and its expression is necessary for a wide range of physiological functions.