Paclitaxel frequently serves as the first-line chemotherapeutic agent for castration-resistant prostate cancer (PCa) patients. However, acquired paclitaxel-resistance almost always occurs after initial responses, and the mechanisms by which this occurs remain largely unknown. The goal of the present study was to identify differentially expressed protein(s) associated with paclitaxel-resistance and further explore the potential mechanisms involved in drug resistance. By comparing the nuclear matrix protein (NMP) patterns of DU145-TxR cells, the previously established stable paclitaxel-resistant PCa cells, with that of the parental DU145 cells using two-dimensional electrophoresis, we found that cytokeratin 18 (CK18) is downregulated in DU145-TxR cells. The downregulation of CK18 in DU145-TxR cells at mRNA, NMP and total cellular protein levels was validated by real-time RT-PCR, immunoblotting and immunofluorescence, indicating that the downregulation of CK18 was a global effect in DU145-TxR cells due to paclitaxel-resistance. Furthermore, in vivo assay of xenograft transplantation confirmed the higher tumorigenicity of DU145-TxR cells, suggesting that these paclitaxel-resistant PCa cells possessed potent cancer stem cell (CSC)-like properties and eventually developed paclitaxel-resistance. Moreover, we determined by immunohistochemistry that CK18 expression in PCa tissues was inversely correlated with tumor grade in a statistically significant fashion, indicating a potential association of the downregulation of CK18 with tumor aggressiveness. Therefore, further study to define the potential role of CK18 may lead to novel therapy strategies as well as clinically useful biomarker for PCa patients.