Various interventions have been proposed to combat the increase of antibiotic resistance and influence antibiotic prescribing practices. A prospective cohort study in a medical intensive care unit was conducted to determine the effect of an antibiotic cycling program on patterns of antibiotic use and to determine patient factors associated with cycling adherence. Four major classes of antibiotics for empirical therapy of suspected gram-negative bacterial infections were rotated at 3- and 4-month intervals. During the study, 1,003 patients received antibiotic therapy with at least one of the study drugs; of the 792 receiving cycle antibiotics during the cycling period, 598 (75.5%) received an on-cycle drug. Compared to the baseline, cycling recommendations increased the use of the target cycle agent: the use of cephalosporins increased during cycle 1 (56 to 64% of total antibiotic days, P < 0.001), fluoroquinolone use increased in cycle 2 (24 to 55%, P < 0.001), carbapenem use increased during cycle 3 (14 to 38%, P < 0.001), and use of extended-spectrum penicillins increased in cycle 4 (5 to 36%, P < 0.001). Overall, 48% of total cycle antibiotic days were compliant with the cycling protocol. On average, 8.8 days per patient were spent receiving on-cycle drugs (range, 1 to 109). Cycle periods that specified carbapenem and fluoroquinolone use had the highest number of off-cycle days (62 and 64%). Predictors of on-cycle antibiotic use were increased severity of illness, as measured by an acute physiology and chronic health evaluation II score, and greater length of intensive care unit stay. In conclusion, the successful implementation of this cycling protocol increased antibiotic heterogeneity over time in the study unit.