Introduction: Inhibition of prostate smooth muscle contraction is an important strategy for medical treatment of lower urinary tract symptoms (LUTS). Besides α1-adrenoceptors, prostate smooth muscle contraction is induced by activation of thromboxane (TXA2) receptors (TXA2-R). Here, we examined the effects of the TXA2-R antagonist, picotamide, on contraction of human prostate tissue. Methods: Prostate tissues were obtained from radical prostatectomy. Effects of picotamide (300 μM), L-665,240 (3 μM), and seratrodast (3 μM) on U46619-, electric field stimulation- (EFS-), phenylephrine-, and noradrenaline-induced contractions were studied in organ baths. Expression of TXA2-R and TXA2 synthase (TXS) was examined by fluorescence stainings. Results: Picotamide, seratrodast, and L-655,240 inhibited concentration-dependent contractions induced by the TXA2 analogue, U46619. Picotamide, but not seratrodast or L-655,240 inhibited frequency-dependent contractions induced by EFS. Picotamide inhibited concentration-dependent contractions induced by noradrenaline, or by the selective α1-adrenoceptor agonist, phenylephrine. In prostate strips, where only submaximal contraction by a low dose of phenylephrine was induced, application of U46619 raised tone to maximum phenylephrine-induced tension. Immunoreactivity for TXA2-R and TXS was observed in the stroma, and in epithelial cells of glands. Colocalization of both immunoreactivites was observed with the smooth muscle markers calponin and α-smooth muscle actin, with the epithelial marker pan-cytokeratin, and with prostate-specific antigen in the stroma and glands. Conclusions: The receptor antagonist picotamide inhibits α1-adrenergic, TXA2-mediated, and EFS-induced contractions in the human prostate. To the best of our knowledge, this is the first antagonist able to inhibit two different contraction systems in the prostate.