Indocyanine green (ICG) is a photothermal agent, photosensitizer, and fluorescence imaging probe which shows specific accumulation in hepatocellular carcinoma (HCC) cells. We recently developed a photodynamic therapy (PDT) using ICG and near-infrared (NIR) laser as a new anti-cancer treatment for HCC. However, the molecular mechanism underlying this effect needs to be elucidated. HuH-7 cells, a well-differentiated human HCC cell line, were transplanted subcutaneously into BALB/c-nu/nu mice for in vivo experiment. ICG was administered 24 h before NIR irradiation. The irradiation was performed at three tumor locations by 823-nm NIR laser on days 1 and 7. The temperature of HuH-7 xenografts increased to 48.5 °C 3 minutes after ICG-NIR irradiation start. Reactive oxygen species (ROS) production was detected after ICG-NIR irradiation both in vitro and in vivo. There was certain anti-tumor effect and ROS production even under cooling conditions. Repeated NIR irradiation increased the cell toxicity of ICG-NIR therapy; the mean tumor volume on day 9 was significantly smaller after ICG-NIR irradiation compared to tumor without irradiation (87 mm3 vs. 1332 mm3; p = 0.01) in HCC mice xenografts model. ICG-NIR therapy induced apoptosis in HCC cells via a photothermal effect and oxidative stress. Repeated ICG-NIR irradiation enhanced the anti-tumor effect.