Purpose Gene transduction using adenoviral vectors is an important research tool. To assess and optimize this technique for glaucoma research, we characterized green fluorescent protein (GFP) expression in the mouse eye after intraocular injection of adenoviral vector encoding GFP (Ad5.CMV-GFP) and evaluated the effect of anti-CD40L antibody administration on GFP expression. Methods Mice were injected with Ad5.CMV-GFP intracamerally (IC) or intravitreally (IVT) with or without anti-CD40L antibody treatment. GFP expression was assessed by in vivo fluorescence intensity with a standardized grading scale. Location of expression was analyzed histologically by fluorescence microscopy. Results Intraocular injection of Ad5.CMV-GFP induced titer-dependent expression of GFP in the anterior segment. In vivo fluorescence was detectable but low after IC injection. After IVT injection, fluorescence in the eye peaked at days 4–7 with a fluorescence grade of 3.0 ±0.0 (mean ±SEM, n=6; injection with 1x108 pfu vector). After day 7, GFP expression declined significantly. Treatment with anti-CD40L antibody increased fluorescence intensity after IC injection, and prolonged GFP expression in the IVT group. At day 43, fluorescence grades of the IVT group were 2.8 ±0.7 (with anti-CD40L) and 1.2 ±0.6 (without antibody). Three-Way ANOVA confirmed that GFP expression was significantly higher in the anti-CD40L than the no antibody group (p=0.013), significantly higher in the IVT than the IC group (p=0.003), and significantly higher in the high viral titer (1x108 pfu) than the low titer (1x107 pfu) group (p=0.010). Fluorescence microscopic examination of cross-sections of eyes indicated GFP expression in the trabecular meshwork (TM), corneal endothelium, and sporadically iris, ciliary body and lens epithelium. Conclusions Intraocular injection of Ad5.CMV-GFP induced GFP expression in the mouse anterior segment, including the TM. Expression was more prominent after IVT injection than IC injection. Anti-CD40L antibody treatment increased both intensity and duration of GFP expression. These findings provide important and practical means to improve duration and efficiency of adenovirus-mediated transgene expression in the eye.