OBJECTIVE To study the association between gastrointestinal colonization of carbapenemase-producing Enterobacteriaceae (CPE) and proton pump inhibitors (PPIs). METHODS We analyzed 31,526 patients with prospective collection of fecal specimens for CPE screening: upon admission (targeted screening) and during hospitalization (opportunistic screening, safety net screening, and extensive contact tracing), in our healthcare network with 3,200 beds from July 1, 2011, through December 31, 2015. Specimens were collected at least once weekly during hospitalization for CPE carriers and subjected to broth enrichment culture and multiplex polymerase chain reaction. RESULTS Of 66,672 fecal specimens collected, 345 specimens (0.5%) from 100 patients (0.3%) had CPE. The number and prevalence (per 100,000 patient-days) of CPE increased from 2 (0.3) in 2012 to 63 (8.0) in 2015 (P<.001). Male sex (odds ratio, 1.91 [95% CI, 1.15-3.18], P=.013), presence of wound or drain (3.12 [1.70-5.71], P<.001), and use of cephalosporins (3.06 [1.42-6.59], P=.004), carbapenems (2.21 [1.10-4.48], P=.027), and PPIs (2.84 [1.72-4.71], P<.001) in the preceding 6 months were significant risk factors by multivariable analysis. Of 79 patients with serial fecal specimens, spontaneous clearance of CPE was noted in 57 (72.2%), with a median (range) of 30 (3-411) days. Comparing patients without use of antibiotics and PPIs, consumption of both antibiotics and PPIs after CPE identification was associated with later clearance of CPE (hazard ratio, 0.35 [95% CI, 0.17-0.73], P=.005). CONCLUSIONS Concomitant use of antibiotics and PPIs prolonged duration of gastrointestinal colonization by CPE. Infect Control Hosp Epidemiol 2016;1418-1425.