Prostate cancer is a leading cause of cancer-related death among men. Early diagnosis and treatment are successful against prostate cancer, yet the clinical treatment of advanced prostate cancer remains a challenge. Gemcitabine is used to treat a broad spectrum of solid tumors; however, the clinical response of prostate cancer patients to gemcitabine is limited. In the present study, we showed that HMGN5, a nucleosome binding protein that can unfold chromatin by binding to histone (H1), is overexpressed in prostate cancer cells and plays an oncogenic role in prostate cancer tumorigenesis and development by activating the MAPK signaling pathway. We also found that sensitivity of prostate cancer cells to gemcitabine was positively correlated with HMGN5 expression. Knockdown of HMGN5 expression reduced the sensitivity of PC-3 cells to gemcitabine, and ectopic HMGN5 expression in DU145 cells enhanced the sensitivity to gemcitabine. Gemcitabine decreased HMGN5 expression, consequently leading to inactivation of the MAPK signaling pathway and cleavage of the PARP protein. Finally, we showed that PC-3 cells acquire gemcitabine resistance by gradual loss of HMGN5 expression. The present study suggests that HMGN5 is a potential biomarker for treating prostate cancer, and patients with a high level HMGN5 will benefit from gemcitabine treatment.