Sulfate is required for detoxification of xenobiotics such as acetaminophen (APAP), a leading cause of liver failure in humans. The NaS1 sulfate transporter maintains blood sulfate levels sufficiently high for sulfonation reactions to work effectively for drug detoxification. In the present study, we identified two loss‐of‐function polymorphisms in the human NaS1 gene and showed the Nas1‐null mouse to be hypersensitive to APAP hepatotoxicity. APAP treatment led to increased liver damage and decreased hepatic glutathione levels in the hyposulfatemic Nas1‐null mice compared with that in normosulfatemic wild‐type mice. Analysis of urinary APAP metabolites revealed a significantly lower ratio of APAP‐sulfate to APAP‐glucuronide in the Nas1‐null mice. These results suggest hyposulfatemia increases sensitivity to APAP‐induced hepatotoxicity by decreasing the sulfonation capacity to metabolize APAP. In conclusion, the results of this study highlight the importance of plasma sulfate level as a key modulator of acetaminophen metabolism and suggest that individuals with reduced NaS1 sulfate transporter function would be more sensitive to hepatotoxic agents. (HEPATOLOGY 2006;43: 1241–1247.)