Aim: To investigate a 1-week once-daily triple therapy with esomeprazole, moxifloxacin, and rifabutin for rescue therapy of Helicobacter pylori infection. Methods: Consecutive patients (n = 103) with at least one previous treatment failure and H. pylori infection resistant to both metronidazole and clarithromycin were treated with esomeprazole 40 mg, moxifloxacin 400 mg, and rifabutin 300 mg, given once daily for 7 days. Eradication was confirmed by histology and culture. CYP2C19 status was determined by polymerase chain reaction-restriction fragment length polymorphism. Results: Intention-to-treat and per-protocol eradication rates were 77.7% (68.4-85.3) and 83.3% (74.4-90.2). Five patients discontinued prematurely (4.8%). Eradication was achieved in 93.1% of poor/intermediate metabolizers and in 78.8% of homozygous extensive metabolizers (p =.14). Eradication rates in patients with one, two, three, and four or more previous failures were 78.3%, 89.6%, 68.6%, and 88.9%, respectively (p =.21). The regimen was effective in seven of nine patients who previously failed quadruple therapy. Post-treatment resistance to moxifloxacin and rifabutin was detected in two (12.5%) and five (31%) patients after treatment failure. Conclusion: Once-daily triple therapy with esomeprazole, moxifloxacin, and rifabutin is a promising, safe, and convenient regimen for rescue therapy of H. pylori infection that may serve as a valuable alternative to quadruple therapy, particularly for patients with intolerance to amoxicillin.