The antimicrobial resistance crisis has persisted despite broad attempts at intervention. Detailed studies of the population dynamics that lead to resistance could identify additional intervention points. An important driver of resistance in the most concerning antibiotic resistant pathogens can be selection imposed on bacterial populations that are not the intended target of antimicrobial therapy. Here we focus on the important nosocomial pathogen Enterococcus faecium in a hospital system where resistance to daptomycin is evolving despite standard interventions. We hypothesized that the intravenous use of daptomycin generates off-target selection for resistance in transmissible gastrointestinal (carriage) populations of E. faecium. We performed a case-control study in which the daptomycin resistance of E. faecium isolated from rectal swabs from daptomycin-exposed patients was compared to a control group of patients exposed to linezolid, a drug with similar indications. In the daptomycin-exposed group, daptomycin resistance of E. faecium from the off-target population was on average 50% higher than resistance in the control group (n=428 independent E. faecium clones from 22 patients). There was also greater phenotypic diversity in daptomycin resistance within daptomycin-exposed patients. Multiple samples over time were available from a subset of patients, and these demonstrate wide variability in temporal dynamics, from long-term maintenance of resistance to rapid return to sensitivity after daptomycin treatment stopped. Our results demonstrate that off-target gastrointestinal populations rapidly respond to intravenous antibiotic exposure. Gastrointestinal populations are the source for faecal transmission and so can be the driver for hospital-wide population level increases in resistance. Focusing on the off-target evolutionary dynamics may offer novel avenues to slow the spread of antibiotic resistance.