In this study, we used nitrogen-doped titanium dioxide (N-TiO2) NPs in conjugation with visible light, and show that both reactive oxygen species (ROS) and autophagy are induced by this novel NP-based photodynamic therapy (PDT) system. While well-dispersed N-TiO2 NPs (≤100 μg/ml) were inert, their photo-activation with visible light led to ROS-mediated autophagy in leukemia K562 cells and normal peripheral lymphocytes, and this increased in parallel with increasing NP concentrations and light doses. At a constant light energy (12 J/cm2), increasing N-TiO2 NP concentrations increased ROS levels to trigger autophagy-dependent megakaryocytic terminal differentiation in K562 cells. By contrast, an ROS challenge induced by high N-TiO2 NP concentrations led to autophagy-associated apoptotic cell death. Using chemical autophagy inhibitors (3-methyladenine and Bafilomycin A1), we confirmed that autophagy is required for both terminal differentiation and apoptosis induced by photo-activated N-TiO2. Pre-incubation of leukemic cells with ROS scavengers muted the effect of N-TiO2 NP-based PDT on cell fate, highlighting the upstream role of ROS in our system. In summary, PDT using N-TiO2 NPs provides an effective method of priming autophagy by ROS induction. The capability of photo-activated N-TiO2 NPs in obtaining desirable cellular outcomes represents a novel therapeutic strategy of cancer cells.