Gallstones are crystalline deposits that form in the gallbladder, often due to bile stasis, dietary factors, and oxidative stress, leading to significant gallbladder dysfunction. This study aimed to evaluate the pathological impact of oxidative stress, lithogenic diets, and antioxidant therapy on gallbladder health and gallstone formation in rabbits. The working hypothesis proposed that gallbladder dyskinesia, linked to pathological wall changes and bile stasis, plays a central role in gallstone formation. Thirty-six healthy local rabbits (10–14 months old, 900–1480 g) were allocated into six groups, each with three males and three females. The control group received a standard diet and water. The H2O2 group was given a standard diet plus 1% hydrogen peroxide in water to induce oxidative stress. The DHC + H2O2 group received 1% dihydrocholesterol (starting week 4) and 1% H2O2 to simulate cholesterol gallstone development. The DHC + vitamin AD3E group followed the same lithogenic diet and were injected with vitamin AD3E (0.1 ml/kg) twice weekly as antioxidant therapy. The cholic + H2O2 group was administered 0.5% cholic acid (from week 4) and 1% H2O2. The cholic + vitamin AD3E group had a similar cholic acid diet along with vitamin AD3E injections. At week six, all rabbits were euthanized, and gallbladders were collected for analysis. The results showed that combining a lithogenic diet with oxidative stress significantly induced gallstone formation and gallbladder damage. Antioxidant therapy with vitamin AD3E offered partial protection, reducing oxidative stress and inflammation. However, 50% of rabbits in the DHC + vitamin AD3E group still developed gallstones, compared to 100% in the DHC + H2O2 group. In conclusion, lithogenic diets combined with oxidative stress accelerate gallstone development and cause epithelial damage, while antioxidant therapy mitigates but does not entirely prevent these effects. Further studies are needed to explore the protective mechanisms of antioxidants.