Accurate recognition of urinary bladder muscularis propria (MP) invasion by urothelial carcinoma is crucial as it is the critical crossroad between conservative and aggressive management for the patient. It is now widely known that an inconsistent layer of muscularis mucosae (MM) muscle exists in the lamina propria, which can mimic the MP muscle, particularly when hyperplastic, making staging extremely challenging in some limited, unoriented, or highly cauterized specimens. Smoothelin is a novel smooth muscle-specific contractile protein expressed only by fully differentiated smooth muscle cells, and not by proliferative or noncontractile smooth muscle cells and myofibroblasts. We performed immunohistochemical staining in the bladder for smoothelin to: (a) evaluate its expression in MM and MP muscle in cystectomy specimens and by comparing the staining pattern with smooth muscle actin (SMA), (b) study MP variations in the bladder trigone and at the ureteric insertion in the bladder wall, and (c) assess the staining pattern of MM and MP in a representative group of transurethral resection of bladder tumor specimens. In contrast to SMA, which equitably stained both types of muscle fibers, smoothelin displayed striking differential immunoreactivity between MM and MP muscle. With smoothelin, the MM muscle (including hyperplastic forms) typically showed absent (19/42, 45%) or weak and focal (18/42, 43%) staining, whereas the MP muscle typically showed strong and diffuse staining (36/42, 86%). Smoothelin accentuated individual muscle fibers within groups of MP bundles only, a feature which was evident in both MM and MP stained by SMA. When only strong and diffuse immunoreactivity in muscle was set as a threshold for positivity, 100% specificity and positive predictive value of smoothelin for MP (vs. MM) was achieved in our study. Smoothelin staining confirmed the morphologic variations in MP muscle in the bladder wall of the trigone and at the ureteric insertion. In addition to the well-defined muscle layers of MM and MP, SMA staining revealed a continuous band of ill-defined haphazardly oriented compact spindle cells that were immediately subjacent to the urothelium in all cases. These spindle cells blended with the morphologically recognizable thin slender fascicles of the MM muscle. We designate this hitherto uncharacterized thin layer of SMA-positive [muscle-specific actin positive (6/6), Masson trichrome stain predominantly blue (5/6)] and smoothelin-negative cells as suburothelial band of myofibroblasts. In all 10 transurethral resection of bladder tumor sections, smoothelin staining was in agreement with the routine light microscopic presence and absence of MP muscle. In conclusion, the relatively distinct immunohistochemical staining pattern of smoothelin between MP and MM (including its hyperplastic forms) makes it a robust and attractive marker to be incorporated in the contemporary diagnostic armamentarium for the sometimes difficult area of staging bladder urothelial carcinoma.