The presence of inflammatory changes and mucopus production in an enterocystoplasty may be similar to the condition of diversion colitis and starvation diarrhea caused by a lack of luminal short-chain fatty acids (SCFAs). We postulate a therapeutic role for intravesical SCFA. Because this treatment will also contact the urothelium, we have assessed the effect on cellular proliferation by utilizing primary urothelial cells in culture. Primary urothelial cells were grown from biopsy samples of normal urothelium obtained intraoperatively. A cocktail of SCFA used in the treatment of diversion colitis was incubated with these cells for time intervals ranging from 30 minutes to 72 hours at drug concentrations ranging from 0.04 to 20 mmol/L butyrate equivalent (BE). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the residual viable biomass to assess growth inhibition. These experiments were repeated on cells grown on matrigel substrate. The human urothelial cancer line RT112 was likewise exposed to SCFAs to assess selectivity between primary and transformed cells. Primary urothelial cells in culture undergo growth inhibition when exposed to SCFAs. The concentration of SCFAs required to reduce the general biomass by 50% or more (IC ≥ 50) was 20 mmol/L BE when exposure was for 2 hours or less. When drug exposure was prolonged for 72 hours, the IC ≥ 50 was 2.5 mmol/L BE. Cells grown on matrigel had their growth similarly inhibited. The IC ≥ 50 for the RT112 cell line was 2.5 mmol/L BE after 72 hours of drug incubation. Primary urothelial cells in culture undergo a time- and dose-dependent growth inhibition when exposed to SCFAs. This inhibition is particularly apparent at the higher doses similar to those in use in clinical practice. Cells grown on a matrigel substrate suffer growth attenuation similar to that affecting cells grown on polystyrene plates. In vivo assessment in a rodent intravesical model is advisable before considering instillations in patients.