Bee venom is a natural mixture and candidate anti-cancer agent with selective cytotoxic effect on some cancer cells. However, the cellular mechanisms of how bee venom selectively targets cancer cells remain elusive. The aim of this study was to reveal the genotoxic effect of bee venom in concordance with the location of β-actin protein throughout the nucleus or/and cytoplasm. For this aim, the level of H2AX phosphorylation (γH2AX) and intracellular location of β-actin were assessed by immunofluorescence in liver (HEPG2) and metastatic breast (MDA-MB-231) cancer cell lines compared to normal fibroblasts (NIH3T3) after bee venom treatment. Colocalisation profiles of γH2AX and β-actin in each cell line were also analysed. The results showed that the levels of γH2AX staining decreased in normal cells but increased in cancer cells. The majority of β-actin was localised within the cytoplasm of normal cells after bee venom treatment, but it was mostly accumulated within the nucleus in cancer cells. Colocalisation of β-actin and γH2AX both in nucleus and cytoplasm was induced in each cancer cell by different patterns. The results showed that normal and cancerous cells had different responses against bee venom, and suggested that bee venom induced a cellular response by the interaction between γH2AX and β-actin.