Background <p>Fluoxetine is commonly used as the initial option for depressive symptoms and other neurological disorders. The repeated use of fluoxetine was related to hepatotoxicity and persistent liver disease. N-acetylcysteine (NAC) and royal jelly, a natural substance, possess hepatoprotective activity.</p> Objective <p>Compare the hepatoprotective activity of N-acetylcysteine and royal jelly against fluoxetine hepatotoxicity in rats.</p> Materials and methods <p>Rats were being randomized to five groups and orally administered every day for 28 days: Control group (0.2 ml of distilled water), fluoxetine group (10 mg/kg), royal jelly (150 mg/kg) + fluoxetine (10 mg/kg) group, NAC (200 mg/kg) + fluoxetine (10 mg/kg) group, and royal jelly (150 mg/kg) + NAC (200 mg/kg) + fluoxetine (10 mg/kg) group. The time between doses was 60 min. After 28-day treatment, the rats were sacrificed after 24 h last dose; blood and liver tissue were collected for the enzymatic and oxidative stress evaluation by ELISA and histological analysis.</p> Results <p>Fluoxetine-induced hepatotoxicity was demonstrated by a significant change (<i>p</i> &lt; 0.05) in the liver enzyme activity and produced hepatic histological abnormalities in tissue compared to the normal control group, while the other groups, N-acetylcysteine, royal jelly, and combination of N-acetylcysteine and royal jelly, all exhibited a significant (<i>p</i> &gt; 0.05) reduction in ALT serum level in contrast to fluoxetine group.</p> Conclusion <p>Royal jelly and N-acetylcysteine are protective against rat hepatotoxicity caused by fluoxetine. This result might result from decreased inflammation.</p>