Historically, intravenous acetylcysteine has been delivered at a fixed dose and duration of 300 mg/kg over 20 to 21 hours to nearly every patient deemed to be at any risk for hepatotoxicity following acetaminophen overdose. We investigated a 12-hour treatment regimen for selected low-risk patients. This was a multicenter, open-label, cluster-controlled trial at six metropolitan emergency departments. We enrolled subjects following single or staggered acetaminophen overdose with normal serum alanine transaminase (ALT) and creatinine on presentation and at 12 hours, and less than 20 mg/L acetaminophen at 12 hours. Patients were allocated to intervention (250 mg/kg over 12-hour) or control (300 mg/kg over 20-hour) regimens by site. The primary outcome was incidence of “hepatic injury” 20 hours following initiation of acetylcysteine treatment, defined as ALT doubling and peak ALT greater than 100 IU/L, indicating the need for further antidotal treatment. Secondary outcomes included incidence of hepatotoxicity (ALT > 1,000 IU/L), peak international normalized ratio (INR), and adverse drug reactions. Of the 449 acetaminophen overdoses receiving acetylcysteine, 100 were recruited to the study. Time to acetylcysteine (median 7 hours [interquartile ratio 6,12] versus 7 hours [6,10]) and initial acetaminophen (124 mg/L [58,171] versus 146 mg/L [66,204]) were similar between intervention and control groups. There was no difference in ALT (18 IU/L [13,22] versus 16 IU/L [13,21]) or INR (1.2 versus 1.2) 20 hours after starting acetylcysteine between groups. No patients developed hepatic injury or hepatotoxicity in either group (odds ratio 1.0 [95% confidence interval 0.02, 50]). No patients represented with liver injury, none died, and 96 of 96 were well at 14-day telephone follow-up. Conclusion: Discontinuing acetylcysteine based on laboratory testing after 12 hours of treatment is feasible and likely safe in selected patients at very low risk of liver injury from acetaminophen overdose.