The aberrant expression of microRNAs (miRNAs) has been found in various types of cancer. The present study found miR-20a to be significantly upregulated in prostate cancer compared with normal prostate tissues. The proliferation and colony formation assays revealed that the downregulation of miR-20a by miR-20a inhibitor suppresses the proliferation of MDA-PCa-2b cells in vitro and also inhibits tumor growth in vivo. Furthermore, a gap junction protein, α 1 (CX43), was identified as a direct target gene of miR-20a. The upregulation of CX43 was detected in MDA-PCa-2b cells after treatment with miR-20a inhibitor both in vitro and in vivo. In conclusion, the findings show that miR-20a significantly contributes to the progression of prostate cancer by targeting CX43.