BACKGROUNDHigh-quality cost estimates for hospital-acquired Clostridium difficile infection (CDI) are vital evidence for healthcare policy and decision-making.OBJECTIVETo evaluate the costs attributable to hospital-acquired CDI from the healthcare payer perspective.METHODSWe conducted a population-based propensity-score matched cohort study of incident hospitalized subjects diagnosed with CDI (those with the International Statistical Classification of Diseases and Related Health Problems, 10th Revision, Canada code A04.7) from January 1, 2003, through December 31, 2010, in Ontario, Canada. Infected subjects were matched to uninfected subjects (those without the code A04.7) on age, sex, comorbidities, geography, and other variables, and followed up through December 31, 2011. We stratified results by elective and nonelective admissions. The main study outcomes were up-to-3-year costs, which were evaluated in 2014 Canadian dollars.RESULTSWe identified 28,308 infected subjects (mean annual incidence, 27.9 per 100,000 population, 3.3 per 1,000 admissions), with a mean age of 71.5 years (range, 0–107 years), 54.0% female, and 8.0% elective admissions. For elective admission subjects, cumulative mean attributable 1-, 2-, and 3-year costs adjusted for survival (undiscounted) were $32,151 (95% CI, $28,192–$36,005), $34,843 ($29,298–$40,027), and $37,171 ($30,364–$43,415), respectively. For nonelective admission subjects, the corresponding costs were $21,909 ($21,221–$22,609), $26,074 ($25,180–$27,014), and $29,944 ($28,873–$31,086), respectively.CONCLUSIONSHospital-acquired CDI is associated with substantial healthcare costs. To the best of our knowledge, this study is the first CDI costing study to present longitudinal costs. New strategies may be warranted to mitigate this costly infectious disease.Infect Control Hosp Epidemiol 2016;37:1068–1078