The epigenetic silencing of tumor suppressor genes (TSGs) is critical in the development of chronic myeloid leukemia (CML).SHP-1functions as a TSG and negatively regulates JAK/STAT signaling. Enhancement ofSHP-1expression by demethylation provides molecular targets for the treatment of various cancers. Thymoquinone (TQ), a constituent ofNigella sativaseeds, has shown anti-cancer activities in various cancers. However, TQs effect on methylation is not fully clear. Therefore, the aim of this study is to assess TQs ability to enhance the expression ofSHP-1through modifying DNA methylation in K562 CML cells. The activities of TQ on cell cycle progression and apoptosis were evaluated using a fluorometric-red cell cycle assay and Annexin V-FITC/PI, respectively. The methylation status ofSHP-1was studied by pyrosequencing analysis. The expression ofSHP-1,TET2,WT1,DNMT1,DNMT3A, andDNMT3Bwas determined using RT-qPCR. The protein phosphorylation of STAT3, STAT5, and JAK2 was assessed using Jess Western analysis. TQ significantly downregulated theDNMT1gene,DNMT3Agene, andDNMT3Bgene and upregulated theWT1gene andTET2gene. This led to hypomethylation and restoration ofSHP-1expression, resulting in inhibition of JAK/STAT signaling, induction of apoptosis, and cell cycle arrest. The observed findings imply that TQ promotes apoptosis and cell cycle arrest in CML cells by inhibiting JAK/STAT signaling via restoration of the expression of JAK/STAT-negative regulator genes.