Photodynamic therapy (PDT) is a promising cancer treatment modality owing to its high spatiotemporal selectivity and noninvasive nature. However, conventional photosensitizers (PSs) used in PDT are responsive only to visible light, which makes them unsuitable for tissue penetration. In this study, we propose a PS based on hot band absorption (HBA), which can be triggered by anti-Stokes light at 808 nm via a one-photon process. The introduction of selenium (Se) into pentamethine cyanine (Secy5) not only facilitates intersystem crossing for reactive oxygen species (ROS) production but also enhances HBA efficiency, thereby prolonging the excitation wavelength. In addition, Secy5 demonstrates excellent biocompatibility, unlike its I-substituted counterpart (Icy5), and produces not only 1O2 but also O2•−, making it a desirable candidate for treating hypoxic solid tumors. According to the results of in vivo and in vitro experiments, Secy5 can efficiently inhibit cancer cell growth via anti-Stokes activation processes, thereby providing a novel approach to design anti-Stokes excitation PSs for anticancer treatment.