Prion protein (PrP) is well studied for its pathogenic role in prion disease, but its potential contribution to other pathological processes is less understood. PrP is expressed in a variety of cancers and at least in pancreatic and breast cancers, its expression appears to be associated with poor prognosis. To understand the role of PrP in breast cancer cells, we knocked down PrP expression in MDA-MB-435 breast cancer cells with small interfering RNA and subjected these cells to a series of analyses. We found that PrP knockdown in these cells does not affect cell proliferation or colony formation, but significantly influences the cellular response to cytotoxic stimuli. Compared to control cells, PrP knockdown cells exhibited an increased susceptibility to serum deprivation induced apoptosis, no change to staurosporine- or paclitaxel-induced cell deaths, and a reduced susceptibility to chemotherapy drug doxorubicin-induced cell death. To understand the mechanism of unexpected role of PrP in exacerbating doxorubicin-induced cytotoxicity, we analyzed cell death related Bcl-2 family proteins. We found that PrP knockdown alters the expression of several Bcl-2 family proteins, correlating with increased resistance to doxorubicin-induced cytotoxicity. Moreover, the enhanced doxorubicin resistance is independent of DNA damage related p53 pathway, but at least partially through the ERK1/2 pathway. Together, our study revealed that silencing PrP in MDA-MB-435 breast cancer cells results in very different responses to various cytotoxic stimuli and ERK1/2 signaling pathway is involved in PrP silencing caused resistance to doxorubicin.