Breast cancer (BC) is the most common malignant tumor in women and the leading cause of cancer deaths worldwide. This work was conducted to estimate the roles of oxidative stress, vitamin B12, homocysteine (HCY), and DNA methylation in BC disease progression. Sixty BC patients (age range 33–80 years) and 30 healthy controls were recruited for this study. Patients with BC were split to group 1 consisted of stage II BC women (low level), and group 2 consisted of patients in stages III and IV (high level). Malondialdehyde (MDA), glutathione peroxidase 3 (GPX3), HCY, and vitamin B12 levels in the study groups were measured. Also, the 5-methylcytosine (5mC) global DNA methylation levels were evaluated. The results showed a significant increase in HCY, and MDA in BC patients compared to healthy controls, with evident increases observed in those with advanced-stage BC (stages III and IV). They were accompanied by significantly reduced levels of 5mC, with a positive correlation between 5mC and the different stages of BC. Also, patients in advanced stages and those with a poor prognosis were exposed to low levels of vitamin B12 and GPX3 (except for the patients in stage IV, which showed increased GPX3 levels). The findings of this study suggest that the differences in global DNA methylation levels at the various phases may be used as a risk factor for developing BC, which indicates the involvement of GPX3 and HCY in BC progression