High doses of non-steroidal anti-inflammatory drugs (NSAIDs) such as acetaminophen may cause oxidative stress and hepatotoxicity. Probiotic bacteria may have beneficial effects in preventing liver damage caused by oxidative stress. The present study aimed to investigate the effects of Lactobacillus rhamnosus on acetaminophen-induced hepatotoxicity. In this study, four groups (n = 6) of rats were used: negative control group, bacterial positive control group, acetaminophen positive control group, and the experimental group. The negative control group did not receive any treatment during the experimental period (21 days). The bacterial control group received 6 × 109 of the bacterial colony forming unit once daily for 21 days. The acetaminophen control group was given a high dose of acetaminophen (1 g/kg) on day 21 of the experiment. The experimental group received the bacterium for 21 days and received a high dose of acetaminophen (1 g/kg) on the last day of the experiment. On day 22, blood samples were taken from all the rats. Then, serum concentrations of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) were measured. The results showed that on day 22 of the experiments, in the acetaminophen group, the concentrations of all the enzymes indicating the hepatotoxicity were higher than the other groups. Pretreatment of Lactobacillus rhamnosus for 21 days could significantly reduce the concentration of the enzymes that indicating acetaminophen toxicity. The administration of the bacterium alone had no significant effect on the serum concentrations of liver enzymes in the serum of rats. Therefore it can be concluded that, pretreatment of lactobacillus rhamnosus may reduce acetaminophen- induced hepatotoxicity.