Propranolol (PL), a non-selective beta-blocker, is a cardiovascular drug widely used to treat hypertension. The present study was concerned with assessing the cytogenetic effects of this drug on Chinese hamster ovary (CHO) cell line. MTT assay was then carried out to determine the cytotoxicity index (IC50) of the drug. The IC50 value of PL was 0.43±0.02 mM. To investigate the clastogenic effects of the drug, chromatid and chromosome breaks and polyploidy in metaphases were analyzed. CHO cells were exposed to different concentrations of the drug (0.1, 0.2, 0.3, 0.4 mM) for 24 hours. Considering that PL has liver metabolism, experiments were carried out in the presence and absence of the metabolic activation system (S9 mix). Mitomycin-C and sodium arsenite were used as positive controls. It was observed that in cells treated with different PL concentrations as 0.1, 0.2 and 0.3 mM, the frequency of chromatid and chromosome breaks as well as polyploidy increased when compared with untreated CHO cells. The addition of S9 mix significantly decreased the chromatid breaks, chromosome breaks and polyploidy compared to the treatment of PL alone. It is concluded that, PL causes chromatid and chromosome aberrations in CHO cell line and the metabolic activation system (S9 mix), playing an important role in drug cytotoxicity reduction.