Background Sepsis treatment has been associated with high costs. Furthermore, both the incidence of sepsis and the severity of illness at presentation appear to be increasing. We estimated healthcare costs related to the treatment of patients with sepsis in the intensive care unit (ICU) and aimed to explain variability in costs between individuals. Methods We performed a prospective cohort study in patients presenting with severe sepsis or septic shock to the ICUs of two tertiary centres in the Netherlands. Resource use was valued using a bottom-up micro-costing approach. Multivariable regression analysis was used to study variability in costs. Results Overall, 651 patients were included, of which 294 presented with septic shock. Mean costs were €2250 (95% CI €2235-€2266) per day and €29,102 (95% CI €26,598-€31,690) per ICU admission. Of the total expenditure, 74% was related to accommodation, personnel, and disposables, 12% to diagnostic procedures, and 14% to therapeutic interventions. Patients with septic shock had higher costs compared with patients with severe sepsis (additional costs: €69 (95% CI €37-€100) per day, and €8355 (95% CI €3400-€13,367) per admission). Site of infection, causative organism, presence of shock, and immunodeficiency were independently associated with costs, but explained only 11% of the total variance. Conclusion Mean costs of sepsis care in the ICU were almost €30,000 per case. As costs were poorly predictable, opportunities for cost savings based on patient profiling upon admission are limited. © 2016, Netherlands Society of Intensive Care. All right reserved.