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Breast cancer, a most common cancer in the women worldwide representing about quarter (25%) of all cancers. The predictions of cancer prevalence show an imperative need for strengthening the present diagnostic/treatment facilities. One such strategy to overcome the treatment burden for the breast cancer is drug repurposing/repositioning. This concept has been growing its importance, the very reason behind this is it decreases significantly the funds needed for the development of therapy for a disease and it increases the chances of the new indications from Phase I to entry in the market. The objective of the study was to evaluated the morphological characteristic, migration features of the cells and cell cycle arrest of the anti-HSV drug Acyclovir against breast cancer cell lines MCF-7 and MDA-MB231. The in vitro cytotoxicity studies, fragmentation assay, long term clonogenic assay and cell cycle analysis were carried out in the study. In MTT assay acyclovir showed IC50 of 3.16 ± 1.10 µg/ml and 3.85 ± 1.54µg/ml respectively with SI of 33.46 tested against MCF-7 and MDAMB-231 cell lines. The platting efficiency was found to be 52.30 % by clonogenic assay. One of the indications of cell death by apoptosis is accumulation of cells in G2/M phase which was observed with Acyclovir in cell cycle analysis. The study showed that Acyclovir inhibited cancer cells propagation, colony formation ability and cell cycle arrest at G2/M phase, while having no effect on the normal cells. These results indicated new insights on the effect of anti-HSV agents on the tumorigenesis and metastasis. [ABSTRACT FROM AUTHOR]