Background: Gabapentin (GP) as an analog of GABA, is the most commonly used medicine to treat neurological pain. Studies revealed continuous application of GP causes bone mass density reduction in patients. The present study aimed to investigate its effect on the osteogenic differentiation of bone marrow mesenchymal stem (BMSCs) which are responsible for producing osteoblasts. Methods and Materials: Rat BMSCs were extracted and treated with different concentrations of GP for 21 days, and then 10-8 and 10-4 M were selected for further study in osteogenic media. Matrix production was investigated using alizarin red quantitative analysis and calcium level estimation. In addition, the activity of alkaline phosphatase (ALP) and expression of osteo-related genes including BMP 2 and 7, RUNX2, osteocalcin, Alp, and collagen 1 were determined. Results: Statistical analysis showed that the matrix production by differentiated BMSCs reduced significantly (p<0.05) in a dose-dependent manner after treatment with GP. In addition, the activity of ALP reduced significantly and the expression of all the investigated genes also reduced significantly (p<0.05) in the presence of GP. Conclusion: GP reduces the osteo-differentiation of BMSCs by affecting related genes which finally causes the reduction of bone matrix production.