Urolithiasis, a condition in which stones are present in the urinary system, including the kidneys and bladder, is a poorly understood yet common disorder worldwide that leads to significant health care costs, morbidity, and work loss. Acetaminophen-induced liver damage is a major cause of death in patients with acute liver failure. Kidney and urinary stones and liver toxicity are disturbances linked to alterations in oxalate and sulfate homeostasis, respectively. The sulfate anion transporter–1 (Sat1; also known as Slc26a1) mediates epithelial transport of oxalate and sulfate, and its localization in the kidney, liver, and intestine suggests that it may play a role in oxalate and sulfate homeostasis. To determine the physiological roles of Sat1, we created Sat1^–/– mice by gene disruption. These mice exhibited hyperoxaluria with hyperoxalemia, nephrocalcinosis, and calcium oxalate stones in their renal tubules and bladder. Sat1^–/– mice also displayed hypersulfaturia, hyposulfatemia, and enhanced acetaminophen-induced liver toxicity. These data suggest that Sat1 regulates both oxalate and sulfate homeostasis and may be critical to the development of calcium oxalate urolithiasis and hepatotoxicity.