Purpose: This study assessed the decrease in intraocular pressure (IOP), the residual outflow resistance of the trabeculum and the filtration site by histology in enucleated pig and human eyes after ab externo trabeculectomy. Method: Measurement of the resistance to aqueous outflow was performed using the constant pressure method before and after ab externo trabeculectomy. Results: The outflow facility was 0.31 ± 0.13 µl/min/mm Hg in pig eyes and 0.24 ± 0.08 µl/min/mm Hg in human eyes before surgery, and 79.0 ± 47.6 µl/min/mm Hg in pig eyes and 6.33 ± 6.67 µl/min/mm Hg in human eyes after ab externo trabeculectomy. On histological examinations the aqueous outflow pathway was seen to be at the level of the residual trabecular meshwork. Conclusion: This study showed that ab externo trabeculectomy significantly lowers IOP and improves outflow facility in enucleated pig and human eyes.