A recent study showed that antlers have evolved a high rate of growth due to the expression of proto-oncogenes and that they have also evolved to express several tumour suppressor genes to control the risk of cancer. This may explain why deer antler velvet (DAV) extract shows anti-tumour activity. The fast growth of antler innervation through the velvet in close association to blood vessels provides a unique environment to study the fast but non-cancerous proliferation of heterogeneous cell populations. We set out to study the anti-cancer effect of DAV in glioblastoma (GB) cell lines in comparison with temozolomide, a chemotherapeutic drug used to treat high-grade brain tumours. Here we report, for the first time, that DAV extract from the tip, but not from mid-parts of the antler, exhibits an anti-tumour effect in GB cell lines (T98G and A172) while being non-toxic in non-cancerous cell lines (HEK293 and HACAT). In T98G cells, DAV treatment showed reduced proliferation (37.5%) and colony-formation capacity (84%), inhibited migration (39%), induced changes in cell cycle progression, and promoted apoptosis. The anticancer activity of DAV extract as demonstrated by these results may provide a new therapeutic strategy for GB treatment.