The aim of this study is to evaluate the effect of the variation of miR-221 on the prostate cancer cells' NE differentiation and invasive function and to examine the function of miR-221 in plasma as a blood-based miRNA biomarker candidate for CaP. The expression of 7 miRNAs in LNCaP, LNCaP-AI, and PC3 prostate cancer cell lines was detected by Northern blotting. LNCaP and LNCaP-AI cells cultured in androgen-depleted medium were transfected with different synthetic miRs. The ability of invasiveness was evaluated by a Matrigel invasion assay. Cell growth was assessed by using the CCK-8 cell proliferation assay at different times. The expression of NSE and DVL2 during the neuroendocrine phenotype and migration were measured by qRT-PCR and Western blot. The level of miR-221 in the prostate cancer samples was measured by qRT-PCR. MiR-221 was significantly increased compared AIPC with ADPC cell lines. Overexpression of miR-221 in LNCaP cells significantly increased the level of NSE expression and induced NE differentiation. Knocking down the level of miR-221 expression with antagonist miR-221 in the LNCaP-AI cell line increased migration and invasion (P < 0.01). DVL2 protein level was up-regulated after transfection of anti-miR-221. MiR-221 was up-regulated in CaP plasma (P < 0.01). We demonstrate a significant difference in miR-221 expression between ADPC and AIPC. MiR-221 may contribute to NE differentiation, which may be the cause for AIPC. We also suggest that miR-221 may control the migration of AIPC cells through DVL2, working as a key regulator in advanced CaP. The role of miR-221 in other target mRNA needs to be further investigated.