Neuroblastoma is one of the most aggressive cancers and has a complex form of differentiation. We hypothesized that the advanced cellular differentiation may alter the susceptibility of neuroblastoma to photodynamic therapy (PDT) and have a selective survival advantage. We compared the photocytotoxicity treated by Hematoporphyrin (Hp) for PDT on human neuroblastoma SH-SY5Y cells with retinoic acid (RA)-differentiated SH-SY5Y cells. The undifferentiated neuroblastoma cells were shown to cause elevated photocytotoxic effect by MTT assay and also confirmed by Annexin V-FITC/PI staining. In undifferentiated cells, Hp-PDT increased the generation of intracellular reactive oxygen species (ROS), the loss of mitochondrial membrane potential, characteristic chromatin condensation displaying, PARP cleavage, the downregulated expression of Bcl-2, and the activation of caspase-9, -3 was more significant than that of the differentiated cells. In undifferentiated SH-SY5Y cells, cell cycle arrest at G2/M phase was accompanied by the decrease in cyclin B1 level, and could be reversed by the disruption of intracellular ROS caused by PDT. Furthermore, the ROS scavenger markedly inhibited Hp-PDT induced activation of caspase-3, a sustained phosphorylation of Akt/GSK-3£] and ERK, and cytotoxicity in undifferentiated SH-SY5Y cells, but not in differentiated SH-SY5Y cells. Blockage of p38 and JNK activation can significantly attenuate PDT-induced viability loss in both SH-SY5Y cells, but the less significant activation of p38 and JNK, as well as more significant phosphorylation of Akt and GSK-3£], and a prolonged ERK activation appeared to make differentiated SH-SY5Y cells more resistant to photocytotoxicity. Collectively, these data suggested that differentiated SH-SY5Y cells were more resistant to PDT induced apoptosis than undifferentiated SH-SY5Y cells, and ROS played the most important regulatory role on the susceptibility to Hp-PDT between undifferentiated and differentiated neuroblastoma cells. These results may have important implications for neuroblastoma patients undergoing PDT.