NKX3.1 is an androgen-regulated homeobox gene that encodes a tissue-restricted transcription factor, which plays an important role in the differentiation of the prostate epithelium. Thus, the role of NKX3.1 as a functional topoisomerase I activity enhancer in cell cycle regulation and the DNA damage response (DDR) was explored in prostate cancer cell lines. As an early response to DNA damage following CPT-11 treatment, we found that there was an increase in the γH2AX(S139) foci number and that total phosphorylation levels were reduced in PC-3 cells following ectopic NKX3.1 expression as well as in LNCaP cells following androgen administration. Furthermore, upon drug treatment, the increase in ATM(S1981) phosphorylation was reduced in the presence of NKX3.1 expression, whereas DNA-PKcs expression was increased. Additionally, phosphorylation of CHK2(T68) and NBS1(S343) was abrogated by ectopic NKX3.1 expression, compared with the increasing levels in control PC-3 cells in a time-course experiment. Finally, NKX3.1 expression maintained a high cyclin D1 expression level regardless of drug treatment, while total γH2AX(S139) phosphorylation remained depleted in PC-3, as well as in LNCaP, cells. Thus, we suggest that androgen regulated NKX3.1 maintains an active DDR at the intra S progression and contributes to the chemotherapeutic resistance of prostate cancer cells to DNA damaging compounds.