Objective: Alteration of the colonic microbiota following antimicrobial exposure allows colonization by antimicrobial-resistant organisms (AROs). Ingestion of a probiotic, such as Lactobacillus rhamnosus GG (LGG), could prevent colonization or infection with AROs by promoting healthy colonic microbiota. The purpose of this trial was to determine the effect of LGG administration on ARO colonization in hospitalized patients receiving antibiotics. Design: Prospective, double-blinded, randomized controlled trial of LGG versus placebo among patients receiving broad-spectrum antibiotics. Setting: Tertiary care center. Patients: In total, 88 inpatients receiving broad-spectrum antibiotics were enrolled. Intervention: Patients were randomized to receive 1 capsule containing 1×1010 cells of LGG twice daily (n = 44) or placebo (n = 44), stratified by ward type. Stool or rectal-swab specimens were collected for culture at enrollment, during admission, and at discharge. Using selective media, specimens were cultured for Clostridioides difficile, vancomycin-resistant Enterococcus spp (VRE), and antibiotic-resistant gram-negative bacteria. The primary outcome was any ARO acquisition. Secondary outcomes included loss of any ARO if colonized at enrollment, and acquisition or loss of individual ARO. Results: ARO colonization prevalence at study enrollment was similar (LGG 39% vs placebo 39%). We detected no difference in any ARO acquisition (LGG 30% vs placebo 33%; OR,1.19; 95% CI, 0.38-3.75) nor for any individual ARO acquisition. There was no difference in the loss of any ARO (LGG 18% vs placebo 24%; OR, 1.44; 95% CI, 0.27-7.68) nor for any individual ARO. Conclusion: LGG administration neither prevented acquisition of ARO nor accelerated loss of ARO colonization.