OBJECTIVES: This study aims to investigate whether thymoquinone (TQ) accelerates fracture healing and modulates inflammatory and regenerative markers in a rat model. MATERIALS AND METHODS: Forty-five male Wistar-Albino rats were randomly divided into seven groups and administered intraperitoneal doses of TQ. A femur fracture was surgically induced, and healing was assessed through radiological, histological, and biochemical analyses. The study included seven groups: a sham-operated control (Group 1), untreated fracture controls at two and four weeks (Groups 2 and 5), and fracture groups treated with TQ at 2.5 or 5 mg/kg/day for two weeks (Groups 3 and 4) or four weeks (Groups 6 and 7). Fracture healing was evaluated at two time points: at the end of two weeks (Groups 1-4) and at the end of four weeks (Groups 5-7). Intraperitoneal administration was chosen to ensure accurate and consistent dosing, as oral administration could lead to variability in bioavailability and potential loss during ingestion. Radiological assessments included measuring total callus diameter, radiopacity ratios, and callus surface area using high-resolution tomography. Histological analysis involved hematoxylin-eosin and immunohistochemical staining. Biochemical analyses measured complete blood count, sedimentation rate, C-reactive protein (CRP), total protein, albumin, calcium, alkaline phosphatase (ALP) and phosphorus. RESULTS: Significant differences in callus diameter were observed between the high dose TQ group (Group 7) and the untreated control (Group 5), while callus area showed significant differences among Groups 5, 6 and 7, and callus surface differed significantly between Groups 3 and 6. In Group 6, total protein and calcium levels increased, while albumin and ALP were lower in Group 7. Histological findings demonstrated that TQ treatment reduced dense fibrous tissue, with initial formation of spongy structures followed by trabecular bone development, indicating enhanced bone formation. Immunohistochemical analysis showed increased interleukin (IL)-10 and proliferating cell nuclear antigen (PCNA) levels, supporting improved fracture healing in high-dose groups. CONCLUSION: Thymoquinone enhanced fracture healing, particularly in the fourth-week groups, improving callus formation, biochemical markers, and histological outcomes.