Botulinum toxin (BTX) is the most lethal naturally occurring toxin known to mankind. Injection of BTX into the urethral sphincter or bladder is an effective treatment for lower urinary tract dysfunction. We reviewed the literature on the mechanisms of action and clinical efficacy of BTX treatment in urologic diseases, with a focus on lower urinary tract dysfunction. Injection of BTX is safe and effective in the treatment of detrusor-sphincter dyssynergia, non-neurogenic pelvic floor spasticity, and refractory overactive bladder. Urodynamic assessment after sphincter injection with BTX reveals a decrease of bladder voiding pressure, urethral pressure profile, and post-void residual urine. An increase of the functional bladder capacity and a decrease of the bladder voiding pressure can be seen after bladder injection with BTX. Clinical improvement was found in a moderate percentage of treated patients in most reported series and lasted for 3 to 14 months without significant adverse effects. In addition, BTX-A treatment inhibits afferent-nerve-mediated bladder contraction. This analgesic effect may expand the application of BTX in the localized genitourinary tract pain syndrome, such as interstitial cystitis and prostatodynia. In conclusion, application of BTX is a promising treatment for lower urinary tract dysfunction with profound basic and clinical implications.