Purpose To compare the effectiveness and differences in slowing myopia progression in Guinea pigs by riboflavin/ultraviolet A (UVA) scleral cross-linking (sCXL) before and after lens-induced myopia (LIM). Methods Forty 4-week-old Guinea pigs were randomly divided into four groups (n = 10 per group): CXL-A, CXL-B, LIM, and Control groups. The right eyes in CXL-A, CXL-B, LIM groups were treated with -10.00 D lenses from 4 to 10-week old and the left eyes were untreated. In CXL-A and CXL-B groups, riboflavin/UVA sCXL was performed on the right eyes at 4 weeks and 8 weeks of age, respectively. Both eyes were untreated in Control group. The intraocular pressure (IOP), the axial length (AXL), and the refraction were measured in vivo at 4, 8, and 10 weeks of age. At 10 weeks of age, the right eyes were enucleated for the tensile test and transmission electron microscopy observations. Results The myopia has been successfully induced in LIM and CXL-B groups during 4-8 weeks. In CXL-A group, the growth rate of AXL and myopic refraction was markedly inhibited during 4-8 weeks and the inhibitory effects diminished during 8-10 weeks. During 8-10 weeks, the growth rate of AXL and myopic refraction in CXL-B were marked suppressed. At 10 weeks of age, the myopia refraction was lower and the AXL was shorter in CXL-A group in comparison to CXL-B group. The IOP was not significantly different among the 4 groups of eyes at 4, 8, and 10 weeks of age. The scleral stiffness, the fibril diameters, and the fibril density of the sclera were significantly increased in CXL-A and CXL-B groups compared to LIM group. Conclusion Riboflavin/UVA sCXL administrated before and after the myopia modeling could both slow the myopia progression in Guinea pigs. The before-myopia preventative sCXL showed lower myopic refraction in the same age comparison between the cross-linked groups. The effect of riboflavin/UVA sCXL might reduce over time and the long-term effect should be further investigated. This sCXL intervention might control the ultrastructure alterations of the sclera during the myopia remodeling.