Prostate smooth muscle contraction is critical for etiology and treatment of male lower urinary tract symptoms (LUTS), and is promoted by small monomeric GTPases (RhoA, Rac). GTPases may be activated by guanosine nucleotide exchange factors (GEFs). GEFs of the cytohesin family may indirectly activate Rac, or ADP ribosolyation factor (ARF) GTPases directly. Here we investigated the expression of cytohesin family GEFs, and effects of the cytohesin inhibitor secinH3 on smooth muscle contraction and GTPase activities in human prostate tissues. Of all four cytohesin isoforms, cytohesin-1 and -2 showed the highest expression in RT-PCR. Western blot and fluorescence staining suggested that cytohesin-2 may be the predominant isoform in prostate smooth muscle cells. Contractions induced by norepinephrine, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analogue U46619, endothelins 1 and 3, as well as neurogenic contractions induced by electric field stimulation (EFS) were reduced by secinH3 (30 µM). Inhibition of EFS-induced contractions appeared to have similar efficacy than inhibition by the α1-adrenoceptor antagonist tamsulosin (300 nM). Combined application of secinH3 plus tamsulosin caused larger inhibition of EFS-induced contractions than tamsulosin alone. Pull down assays demonstrated inhibition of the small monomeric GTPase ARF6 by secinH3, but no inhibition of RhoA or Rac1. In conclusion, we suggest that a cytohesin/ARF6 pathway takes part in smooth muscle contraction. This may open attractive new possibilities in medical treatment of male LUTS.