OBJECTIVE: We tested the hypothesis that hypercapnic acidosis is associated with reduced mortality rate in patients with acute lung injury independent of changes in mechanical ventilation. DESIGN: Secondary analysis of randomized clinical trial data using hypothesis-driven multivariate logistic regression. SETTING: Randomized, multiple-center trial (n = 861) comparing 12 mL/kg to 6 mL/kg predicted body weight tidal volumes previously published by the National Institutes of Health Acute Respiratory Distress Syndrome (ARDS) Network. PATIENTS: Acute lung injury patients enrolled in a randomized, multiple-center trial (n = 861). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The adjusted odds ratio and 95% confidence intervals (CI) for 28-day mortality rate associated with hypercapnic acidosis defined as day 1 pH <7.35 and Pa(CO2) >45 mm Hg were 0.14 (95% CI 0.03-0.70, p = .016) in the 12 mL/kg predicted body weight tidal volume group and 1.18 (95% CI 0.59-2.35, p = .639) in the 6 mL/kg predicted body weight tidal volume group. Other definitions of hypercapnic acidosis spanning a range of magnitudes suggest a dose-response association between hypercapnic acidosis and 28-day mortality in the 12 mL/kg predicted body weight tidal volume group. None of our definitions of hypercapnic acidosis were associated with reduction in 28-day mortality in the 6 mL/kg predicted body weight tidal volume group. CONCLUSIONS: Hypercapnic acidosis was associated with reduced 28-day mortality in the 12 mL/kg predicted body weight tidal volume group after controlling for comorbidities and severity of lung injury. These results are consistent with a protective effect of hypercapnic acidosis against ventilator-associated lung injury that was not found when the further ongoing injury was reduced by 6 mL/kg predicted body weight tidal volumes.