Objective:To evaluate the Orange County Clostridium difficile infection (CDI) prevention collaborative’s effect on rates of CDI in acute-care hospitals (ACHs) in Orange County, California.Design:Controlled interrupted time series.Methods:We convened a CDI prevention collaborative with healthcare facilities in Orange County to reduce CDI incidence in the region. Collaborative participants received onsite infection control and antimicrobial stewardship assessments, interactive learning and discussion sessions, and an interfacility transfer communication improvement initiative during June 2015–June 2016. We used segmented regression to evaluate changes in monthly hospital-onset (HO) and community-onset (CO) CDI rates for ACHs. The baseline period comprised 17 months (January 2014–June 2015) and the follow-up period comprised 28 months (September 2015–December 2017). All 25 Orange County ACHs were included in the CO-CDI model to account for direct and indirect effects of the collaborative. For comparison, we assessed HO-CDI and CO-CDI rates among 27 ACHs in 3 San Francisco Bay Area counties.Results:HO-CDI rates in the 15 participating Orange County ACHs decreased 4% per month (incidence rate ratio [IRR], 0.96; 95% CI, 0.95–0.97; P < .0001) during the follow-up period compared with the baseline period and 3% (IRR, 0.97; 95% CI, 0.95–0.99; P = .002) per month compared to the San Francisco Bay Area nonparticipant ACHs. Orange County CO-CDI rates declined 2% per month (IRR, 0.98; 95% CI, 0.96–1.00; P = .03) between the baseline and follow-up periods. This decline was not statistically different from the San Francisco Bay Area ACHs (IRR, 0.97; 95% CI, 0.95–1.00; P = .09).Conclusions:Our analysis of ACHs in Orange County provides evidence that coordinated, regional multifacility initiatives can reduce CDI incidence.