β-thalassemia is a group of hereditary, autosomal recessive blood disorders caused by reduced synthesis or absence of β-globin chains. Reactivation of hemoglobin F (Hb-F) by pharmacological means is an excellent remedial approach for treating β-thalassemia. Hydroxyurea is currently the only approved drug for Hb-F induction therapy. However, the variable efficacy and high cytotoxicity concerns of hydroxyurea have limited its utilization in clinical practices. This preclinical investigation demonstrated the potential of the antiviral drug entecavir (ETV) as an Hb-F inducer using the K562 cell line and validated in a β-YAC transgenic mice model . In the K562 cell line and β-YAC transgenic mice, the effects of ETV on Hb-F induction were investigated in a dose dependent manner. In mice the dose was given orally for 6 days/week for 4 weeks. A network pharmacology-based approach and molecular docking were also utilized to predict the target genes and the underlying mechanism of ETV in Hb-F induction. ETV in a dose dependent significantly increased the level of total haemoglobin, gamma- globin mRNA expression, and Hb-F production in the K562 cell line. In β-YAC mice, ETV confirmed gamma-globin gene expression at the mRNA and protein levels, as evidenced by increased haemoglobin F production in red blood cells. Furthermore, the docking analysis indicated that NFKB, PTGS2, HDAC2, and HIFIA are potential key targets of ETV, possibly involved in Hb-F induction, and must be validated experimentally. ETV, an approved drug for antiviral activity, increased the level of Hb-F in K562 cell line and in β-YAC transgenic mice, may suggest its use for the management of β-thalassemia. However, further research is required to test the drug's efficacy and the underlying gene targets in CD34 ⁺ erythroid progenitors of β-thalassemic patients.