Protoporphyrin IX (PpIX) is an effective hematoporphyrin derivative, widely adopted in photodynamic therapy (PDT) and sonodynamic therapy (SDT). As a sensitizer, PpIX could significantly enhance laser light or ultrasound causing tumor cell damage in PDT/SDT studies. However, the biological function of PpIX itself has not been carefully defined. Recently, studies indicate that PpIX alone can inhibit Hela cell proliferation, but the potential mechanism was unclear. Therefore, in the present study it was investigated whether the proliferation inhibition effect generally occurred in human breast cancer MCF-7 and MDA-MB-231 cells. Different sensitivities and the involved mechanisms were carefully explored. Our results show that PpIX preferentially accumulated and selectively caused cell damage in human breast cancer MCF-7 and MDA-MB-231 cells compared with mouse embryonic fibroblast NIH-3T3. In vitro, PpIX induced cell viability decrease, intracellular ROS (reactive oxygen species) generation, and DNA damage in a concentration-dependent and cell-specific manner. MCF-7 was more sensitive to PpIX than MDA-MB-231 cells at the same PpIX dose. Western blots showed obvious enhancement of P53, and PUMA in a concentration dependent manner in MCF-7 cells, but not in MDA-MB-231 cells. In cell-free system, we also found that PpIX could interact with some large biological molecules, such as calf thymus DNA, and induce hyperchromic effects in spectroscopic analysis. Our findings imply that DNA might be one of the main targets of PpIX, and PpIX alone can cause significant tumor cell damage through ROS generation, while P53 status may play an important role in these processes.