Aberrantly expressed microRNAs (miRs) are extensively involved in tumorigenesis. microRNA-340 (miR-340) has been reported as a tumor suppressor in various cancer types. However, whether miR-340 plays an important role in prostate cancer remains unknown. This study aims to investigate the expression pattern of miR-340 and its functional significance in prostate cancer. Results showed that miR-340 expression was frequently downregulated in human prostate cancer cell lines and cancer tissues. miR-340 overexpression suppressed proliferative and invasive properties of prostate cancer cells. This overexpression also promoted prostate cancer cell apoptosis. Conversely, miR-340 silencing showed an opposite effect. Intriguingly, on the basis of bioinformatics analysis and luciferase reporter assay, we found that miR-340 directly targeted the 3'-untranslated region of the high-mobility group nucleosome-binding domain 5 (HMGN5). Quantitative polymerase chain reaction and western blot analysis further verified the results and demonstrated that miR-340 regulated HMGN5 expression. Correlation analysis also showed that HMGN5 expression levels were significantly inversely correlated with the miR-340 expression in prostate cancer tissues. Furthermore, miR-340 overexpression significantly decreased the protein expression of cyclin B1, Bcl-2, and matrix metalloproteinase-9, which are critical regulators for maintaining tumorigenic potential of cancer cells. In addition, overexpression of HMGN5 significantly reversed the inhibitory effect of miR-340 on prostate cancer cell proliferation and invasion. In summary, this study suggests that miR-340 suppresses the tumorigenic potential of prostate cancer cells. Moreover, the decreased miR-340 expression may contribute to the development and progression of prostate cancer through a mechanism that involves HMGN5. Thus, miR340 and its target gene HMGN5 can serve as potentially useful therapeutic candidates for prostate cancer treatment.