Helicobacter hepaticus and Helicobacter pylori both belong to Helicobacter species. Strains of Lactobacillus acidophilus, including L4 and L6, have shown significant inhibitory effects on H. pylori. Based on this phenomenon, we aim to investigate the inhibitory effect of L. acidophilus on H. hepaticus. Both standard and isolated H. hepaticus strains were grown under microaerophilic conditions at 37 °C in the presence of L. acidophilus supernatant, or lactic acid. The diameters of the inhibition zones were measured on the solid culture media. In liquid culture, the cell concentrations were measured and the urease activity was determined by phenol red staining. Sixteen strains of L. acidophilus isolated from human feces (named as L1-L16) showed anti-H. hepaticus effects. Two of them (L4 and L6) exhibited the most apparent effects on H. hepaticus inhibition. The L. acidophilus supernatant of L4 and L6 significantly increased the diameters of the inhibition zones compared with that of the lactic acid control (P < 0.05). The inhibitory role of L. acidophilus supernatant was independent of the pH value of solution (P > 0.05). Moreover, in liquid culture, L. acidophilus supernatant significantly reduced the cell growth rate and the urease activity of H. hepaticus cells in a time-dependent pattern (P < 0.05 compared with lactic acid control). No obvious difference was observed between the standard and isolated strain of H. hepaticus (P > 0.05). Our results indicate that L. acidophilus can decrease the viability and urease activity of H. hepaticus in vitro and this inhibition is independent of pH levels. This provides evidence for developing novel approaches for the prevention and treatment of H. hepaticus infection.