ObjectiveTo evaluate the effects of latanoprostene bunod on intraocular pressure (IOP) and pupil diameter (PD) in normal cats and cats with feline congenital glaucoma (FCG).Animals StudiedFive normal and 5 FCG cats.ProceduresThis masked, controlled crossover study comprised a 1‐day Pre‐treatment phase followed by two 10‐day Treatment phases, each followed by a 10‐day Recovery phase. During treatment, all cats received twice daily 0.005% latanoprost (LAT) or 0.024% latanoprostene bunod (LBN) in a randomized eye. Following Recovery, the same eye was treated with the opposite drug. Contralateral eyes served as saline‐treated controls. Intraocular pressure and PD measurements were performed three times daily during all study phases. Data were analyzed via constrained longitudinal data analysis models.ResultsNeither drug significantly reduced IOP in normal cats. In FCG cats, statistically significant reductions in mean (95% CI) IOP were observed relative to controls 4 h after LAT and LBN treatment (−5.5 mmHg [−8.4, −2.5], p < .001, −7.2 mmHg [−10.2, −4.3], p < .001, respectively). These differences represented 28.4% and 37.9% IOP reductions, respectively. Mean IOP reduction after 4 h was significantly greater with LBN treatment compared to LAT (−1.8 mmHg [−3.2, −0.4], p = .012). However, these IOP reductions were not considered clinically significant. Both drugs similarly reduced PD in normal and FCG cats.ConclusionsTransient IOP reduction was observed after topical administration of LAT and LBN in FCG cats; and mean IOP difference was statistically significantly greater in LBN‐treated eyes. However, the apparent enhanced hypotensive effect of LBN is not clinically significant.