Background: Bone marrow-derived mesenchymal stem cells (BMMSCs) transplantation has been considered as a promising milestone in liver fibrosis treatment. However, low amounts of homing are a major obstacle. We aimed to investigate the role of melatonin pretreatment in BMMSC homing into experimental liver fibrosis. Methods: BMMSCs were obtained, grown, propagated and preconditioned with 5 µM melatonin and analyzed for multipotency and immunophenotypic features at passage three. The cells were labelled with CM-Dil and infused into the rats received the i.p. injection of carbon tetrachloride (CCl₄) for five weeks to induce liver fibrosis. Animals were divided into two groups: One group received BMMSCs, whereas the other group received melatonin-pretreated BMMSCs (MT-BMMSCs). After cell injection at 72 h, animals were sacrificed, and the liver tissues were assessed for further evaluations: fibrosis using Masson's trichrome and hematoxylin and eosin staining and homing using fluorescent microscopy and flow cytometry. Results: BMMSCs and MT-BMMSCs expressed a high level of CD44 but low levels of CD11b, CD45 and CD34 (for all P≤0.05) and were able to differentiate into adipocytes and Schwann cells. CCl₄ induction resulted in extensive collagen deposition, tissue disruption and fatty accumulation with no obvious difference between the two groups. There was a significant increase in homing of MT-BMMSCs in both florescent microscopy (P≤0.001) and flow cytometry (P≤0.01) assays, as compared with non-treated BMMSCs. Conclusion: This study indicates the improved homing potential of BMMSCs in pretreatment with melatonin. Therefore, this strategy may represent an applied approach for improving the stem cell therapy of liver fibrosis.