Purpose <p>The β₃-agonist vibegron is available for treatment of storage symptoms, but in vitro data in human tissues are not yet available. Based on findings with mirabegron, mechanisms other than inhibition of voiding contractions in the detrusor may account for symptom improvements, and off-target effects in the prostate and bladder appear possible. Here, we examined vibegron effects in human detrusor and prostate tissues.</p> Methods <p>Detrusor and prostate tissues were obtained from radical cystectomy, radical prostatectomy and laser enucleation. Concentration response curves for agonists and frequence response curves for electric field stimulation (EFS) were examined in organ baths with 0.01, 0.1, 1 and 10&#xa0;µM vibegron or vehicle control.</p> Results <p>In detrusor tissues, 10&#xa0;µM vibegron reduced EFS-induced contractions by half, while no biologically relevant inhibitions occurred with lower concentrations or with carbachol- or methacholine-induced contractions. Pretensions before contractions were relaxed &gt; 20% by 0.1–10&#xa0;µM vibegron. EFS-induced contractions were halved by 10&#xa0;µM vibegron in prostatectomized tissues, but unaffected by lower concentrations and in laser-enucleated prostate tissues. Additionally, 10&#xa0;µM vibegron right-shifted concentration response curves and increased the EC₅₀ values for noradrenaline, phenylephrine and methoxamine in prostatectomized and laser-enucleated prostate tissues, while lower concentrations had no consistent effect.</p> Conclusions <p>Similar to mirabegron, improvements of storage symptoms by vibegron involve mechanisms beyond inhibition of voiding contractions. Off-target effects occur with 10&#xa0;µM, and include inhibition of neurogenic contractions and antagonism of prostatic α_1A-adrenoceptors. Vibegron effects in the prostate may differ between patients with low BPH progression, and patients needing surgery for BPH.</p>