Identifying biomarkers and signaling pathways are important for the management of prostate cancer (CaP) radioresistance. In this study, we identified differential proteins and signaling pathways from parental CaP cell lines and CaP radioresistant (RR) sublines using a label-free LC-MS/MS proteomics approach. A total of 309 signaling pathway proteins were identified to be significantly altered between CaP and CaP-RR cells (p ≤ 0.05, fold differences >1.5, ≥80% power). Among these proteins, nineteen are common among three paired CaP cell lines and associated with metastasis, progression and radioresistance. The PI3K/Akt, VEGF and glucose metabolism pathways were identified as the main pathways associated with CaP radioresistance. In addition, the identified potential protein markers were further validated in CaP-RR cell lines and subcutaneous (s.c) animal xenografts by western blotting and immunohistochemistry, respectively and protein aldolase A (ALDOA) was selected for a radiosensitivity study. We found the depletion of ALDOA combined with radiotherapy effectively reduced colony formation, induced more apoptosis and increased radiosensitivity in CaP-RR cells. Our findings indicate that CaP radioresistance is caused by multifactorial traits and downregulation of ALDOA increases radiosensitivity in CaP-RR cells, suggesting that controlling these identified proteins or signaling pathways in combination with radiotherapy may hold promise to overcome CaP radioresistance.