Mast cells and degranulation of pre-formed inflammatory mediators contribute to lower urinary tract symptoms. This study investigates pathways by which the mast cell stimulator Compound 48/80 alters urinary bladder smooth muscle contractility via mast cell activation. We hypothesized that: (1) mast cell degranulation will cause spontaneous urinary bladder smooth muscle contractions; and (2) these contractions are caused by urothelium-derived PGE2. Urothelium intact and denuded urinary bladder strips were collected from mast cell sufficient (C57Bl/6) and deficient (B6.Cg-Kitw-sh) mice to determine if Compound 48/80 altered urinary bladder smooth muscle (UBSM) contractility. Electrical field stimulation was used to assess the effects of Compound 48/80 on nerve-evoked contractions. Antagonists/inhibitors were utilized to identify prostanoid signaling pathways activated or if direct activation of nerves was involved. Compound 48/80 caused slow-developing contractions, increased phasic activity, and augmented nerve-evoked responses in both mast cell sufficient and deficient mice. Nerve blockade had no effect on these responses; however, they were eliminated by removing the urothelium. Blocking P2 purinoreceptors, cyclooxygenases, or G protein signaling abolished Compound 48/80 responses. However, only combined blockade of prostaglandin E2 (EP1), prostaglandin F2α (FP), and thromboxane A2 (TP) receptors inhibited Compound 48/80 induced responses. Thus, the effects of compound 48/80 are urothelium dependent, but independent of mast cells. Further, these effects are mediated by druggable inflammatory pathways that may be used to manage inflammatory non-neurogenic bladder hyperactivity. Finally, these data strongly suggest that great care must be taken when using Compound 48/80 to determine mast cell-dependent responses in the urinary bladder.