Primula vulgaris belongs to the genus Primula, members of which are frequently used in folk medicine. Various studies have investigated the cytotoxic effect of different Primula species, but there have been limited studies on the cytotoxic effect of P. vulgaris. The aim of this study was to investigate the cytotoxic effects, and possible mechanisms involved, of P. vulgaris flower extract on human cervical cancer (HeLa) cells. The cytotoxic effect of the extract on HeLa cells was revealed using the MTT assay. Mechanisms involved in the extract's cytotoxic effect were then investigated in terms of apoptosis, mitochondrial membrane potential, and the cell cycle, using fluorometric methods. P. vulgaris flower extract exhibited selective cytotoxic effects against HeLa cells by arresting their cell cycle at the S phase, and inducing the number of apoptotic cells compared to normal fibroblast cells by reducing mitochondrial membrane potential in a concentration-dependent manner. This is the first study to reveal the antiproliferative effect of P. vulgaris flower extract. Further studies are now needed to identify the cytotoxic molecules in the extract and their mechanisms.