Background While numerous studies have confirmed ATP's importance in bladder physiology/pathophysiology, the literature is still conflicted regarding the mechanism of ATP release from the urothelium. Multiple mechanisms have been identified including non‐vesicular release via pannexin channels as well as vesicular release via a mechanism blocked by botulinum toxin. Recently, it has been shown that lysosomes contain significant stores of ATP which can be released extracellularly in response to Toll‐like receptor (TLR) stimulation. Objective The goal of the current study was to determine if lysosomal exocytosis occurs in urothelial cells in response to TLR4 stimulation by its agonist, bacterial lipopolysaccharide (LPS). Materials and Methods Human urothelial cells from an immortalized cell line (TRT‐HU1) were treated with bacterial LPS (100 μg/ml) or the nicotinic agoinist cytisine (100 μM) and extracellular release of ATP and lysosomal acid phosphatase were measured. Pannexin‐mediated ATP release and lysosomal ATP release were differentiated using Brilliant Blue FCF to inhibit pannexin channels and glycyl‐l‐phenylalanine‐β‐naphthylamide (GPN) to destroy lysosomes. The mechanisms controlling lysosomal exocytosis were examined using lysosomal pH measurements using LysoSensor dye and intracellular calcium signaling using Fura‐2. Results Stimulation of TRT‐HU1 cells with LPS significantly increased ATP release, which was inhibited by GPN, but not by Brilliant Blue FCF. Conversely, stimulation with cytisine induced ATP release that was sensitive to Brilliant Blue FCF but not GPN. LPS stimulation also induced the release of the lysosomal acid phosphatases. LPS increased lysosomal pH and direct alkalization of lysosomal pH using chloroquine or bafilomycin A1 induced ATP and acid phosphatase release, indicating an important role for pH in lysosomal exocytosis. Additionally, stimulation of lysosomal transient receptor potential mucolipin 1 calcium channels evoked intracellular calcium transients as well as ATP release. Conclusion These data indicate that LPS‐induced ATP release from urothelial cells is mediated by lysosomal exocytosis, a vesicular mechanism distinctly separate from non‐vesicular release via pannexin channels.