Polymyxin B hemoperfusion reduces blood endotoxin levels in sepsis. Endotoxin activity can be measured in blood with a rapid assay. Treating patients with septic shock and elevated endotoxin activity using polymyxin B hemoperfusion may improve clinical outcomes. To test whether adding polymyxin B hemoperfusion to conventional medical therapy improves survival compared with conventional therapy alone among patients with septic shock and high endotoxin activity. Multicenter, randomized clinical trial involving 450 adult critically ill patients with septic shock and an endotoxin activity assay level of 0.60 or higher enrolled between September 2010 and June 2016 at 55 tertiary hospitals in North America. Last follow-up was June 2017. Two polymyxin B hemoperfusion treatments (90-120 minutes) plus standard therapy completed within 24 hours of enrollment (n = 224 patients) or sham hemoperfusion plus standard therapy (n = 226 patients). The primary outcome was mortality at 28 days among all patients randomized (all participants) and among patients randomized with a multiple organ dysfunction score (MODS) of more than 9. Among 450 eligible enrolled patients (mean age, 59.8 years; 177 39.3% women; mean APACHE II score 29.4 range, 0-71 with higher scores indicating greater severity), 449 (99.8%) completed the study. Polymyxin B hemoperfusion was not associated with a significant difference in mortality at 28 days among all participants (treatment group, 84 of 223 37.7% vs sham group 78 of 226 34.5%; risk difference RD, 3.2%; 95% CI, -5.7% to 12.0%; relative risk RR, 1.09; 95% CI, 0.85-1.39; P = .49) or in the population with a MODS of more than 9 (treatment group, 65 of 146 44.5% vs sham, 65 of 148 43.9%; RD, 0.6%; 95% CI, -10.8% to 11.9%; RR, 1.01; 95% CI, 0.78-1.31; P = .92). Overall, 264 serious adverse events were reported (65.1% treatment group vs 57.3% sham group). The most frequent serious adverse events were worsening of sepsis (10.8% treatment group vs 9.1% sham group) and worsening of septic shock (6.6% treatment group vs 7.7% sham group). Among patients with septic shock and high endotoxin activity, polymyxin B hemoperfusion treatment plus conventional medical therapy compared with sham treatment plus conventional medical therapy did not reduce mortality at 28 days. ClinicalTrials.gov Identifier: NCT01046669.