We have previously demonstrated that a collagen matrix derived from lamina propria, commonly known as bladder submucosa (BSM matrix), is a suitable biomaterial for several urologic applications, including reconstruction of the bladder and urethra in experimental models and clinical trials. In the present study, we evaluated the physical properties of BSM as well as its biocompatibility, cellular interactions, and ability to support the formation of functional tissue in order to determine whether this biomaterial could serve as a matrix for urinary smooth muscle tissue engineering. BSM matrix resembles the extracellular matrix of bladder submucosa in its native structure, composition, and mechanical properties. BSM matrix supported normal mitochondrial metabolic and proliferative functions of human urinary smooth muscle cells and did not induce cytotoxic effects in vitro. When implanted in vivo, BSM matrix promoted the regeneration of urinary smooth muscle tissues with contractility, which is a smooth muscle-specific tissue function. These results suggest that BSM matrix would be a useful biomaterial for urinary smooth muscle reconstruction.