Hypericin (HYP)-mediated photodynamic therapy (PDT) is a new alternative treatment strategy for colon cancer inducing various cell-death pathways. Apoptotic cell death is the desired cellular fate in cancer cells. Therefore, we investigated the apoptotic pathways by determining apoptosis-related proteins (survivin, caspase-3, caspase-9, Bcl-2, and Bax) at the mRNA level using real-time polymerase chain reaction (qPCR), and the percentage of survivin was determined by survivin ELISA in HT-29 and Caco-2 colon cancer cell lines. The downregulation of survivin was significant 16 h after PDT for both cells, while caspase-3 upregulation was apparent 24 h after PDT. Caspase-9 and caspase-3 upregulations were parallel to each other. There was no Bcl-2 expression in HT-29 cells, but we observed downregulation in Bcl-2 expression in Caco-2 cells after HYP photoactivation. The Bax expression downregulated significantly after 24 h incubation in HT-29 cells, while it was upregulated after 16 h incubation in Caco-2 cells. The present study provides evidence that HYP-induced alterations in apoptosis-related protein expression ended in different responses in HT-29 and Caco-2 colon cancer cells, and these may be used in the treatment of chemotherapy-resistant cancer types.