Current knowledge suggests that infection by carbapenem-resistant enterobacteria is preceded by gut colonization. It is hypothesized that colonization is eradicated by non-absorbable antibiotics like rifaximin. We investigated the effect of rifaximin against carbapenem-resistant Klebsiella pneumoniae (CRKP) in vitro and in a mouse model. We studied the in vitro efficacy of rifaximin against 257 CRKP clinical isolates, 188 KPC producers and 69 OXA-48 producers, by minimum inhibitory concentration and time-kill assays. We then developed a model of gut colonization by feeding 30 C57Bl6 mice with 108 cfu of one KPC-KP isolate for 7 days; mice were pre-treated orally with saline, omeprazole or ampicillin. Then, another 60 mice with established KPC-2 gut colonization received orally for 7 consecutive days rifaximin 180 mg/kg dissolved in ethanol and 4% bile or vehicle. On days 0, 3 and 7 stool samples were collected; mice were sacrificed for determination of tissue outgrowth. At a concentration of 1000 μg/ml rifaximin inhibited 84.8% of CRKP isolates. Α 3 × log10 decrease of the starting inoculum was achieved by 100, 250 and 500 μg/ml of rifaximin after 24 h against 25, 55 and 55% of isolates. Pre-treatment with ampicillin was necessary for gut colonization by KPC-KP. Treatment with rifaximin succeeded in reducing KPC-KP load in stool and in the intestine. Rifaximin inhibits at clinically meaningful gut concentrations the majority of CRKP isolates and is efficient against gut colonization by KPC-KP.