<i>Background:</i> Specific strains of <i>Lactobacillus acidophilus</i> are known to inhibit intestinal cell adhesion and invasion by enterovirulent bacteria. As <i>L. acidophilus</i> can survive transiently in the human stomach, it may downregulate <i>Helicobacter pylori</i> infection. <i>Methods:</i> The ability of <i>L. acidophilus (johnsonii)</i> La1 supernatant to interfere with <i>H. pylori </i>bacterial growth, urease activity, and adhesion to epithelial cells was tested in vitro. Its effect on <i>H. pylori</i> infection in volunteers was monitored in a randomized, double-blind, controlled clinical trial, using a drinkable, whey-based, La1 culture supernatant. <i>H. pylori </i>infected volunteers were treated 14 days with 50 ml of La1 supernatant four times a day combined with either omeprazole 20 mg four times a day or with placebo. Infection was assessed by breath test, endoscopy, and biopsy sampling, performed at inclusion, immediately at the end of the treatment (breath test only), and 4 weeks after the end of the treatment. <i>Results:</i> La1 supernatant inhibited <i>H. pylori</i> growth in vitro, regardless of previous binding of <i>H. pylori</i> to epithelial cells. In 20 subjects (8 females, 12 males, mean age 33.1 years) a marked decrease in breath test values was observed immediately after treatment with La1 supernatant, both in the omeprazole and in the placebo group (median 12.3 vs. 28.8 and 9.4 vs. 20.4, respectively; p < 0.03). In both treatment groups, breath test values remained low 6 weeks after treatment (omeprazole treated 19.2, placebo treated 8.3; p < 0.03 vs. pretreatment), but the persistence of <i>H. pylori</i> infection was confirmed in gastric biopsies. <i>Conclusion:</i> La1 culture supernatant shown to be effective in vitro has a partial, acid-independent long-term suppressive effect on <i>H. pylori</i> in humans.