PURPOSE: To evaluate the biomechanical difference of human scleral collagen cross-linking (CXL) by comparing different riboflavin-instilling methods and different crosslinked regions (equatorial and posterior sclera). METHODS: Fifteen donor human eyes were randomly divided into five groups. One group, in which CXL was not applied, was designated as the control group. In the remaining four groups, 0.1% riboflavin solution was instilled on the scleral surface for 5, 10, 20, or 30 minutes, respectively, followed by 30 minutes of ultraviolet A irradiation. The equatorial and posterior scleral strips in each eye were dissected. Stress-strain measurements of all scleral strips were performed by a biomaterial tester. Young modulus was calculated at 8% strain. Data of the stress and Young modulus in different regions and groups were compared using one-way analysis of variance. RESULTS: Under a 1 mm/minute stretching, the sclera exhibited an exponential stress-strain behavior. The stress and modulus of equatorial and posterior sclera after CXL gradually increased with riboflavin instillation before surgery. No statistical difference was noted in the modulus between 20 and 30 minutes riboflavin infiltration after CXL (P >.05). At the same strain levels, equatorial sclera with and without CXL exhibited higher stress and Young modulus than that of posterior sclera. CONCLUSIONS: Equatorial and posterior human sclera may be enhanced by CXL with riboflavin/ultraviolet A irradiation. Equatorial scleral CXL may be a good choice for the treatment of progressive myopia. Because of its safety and efficacy, 20 minutes of riboflavin infiltration before CXL is recommended. [J Refract Surg. 2012;28(10):723-728.] doi:10.3928/1081597X-20120921-08