Purpose The purpose of the study was to evaluate myopia progression and axial elongation after stopping 0.01% atropine eye drops through a 2-year cross-over study. Methods This study was a randomized, double-masked, placebo-controlled, cross-over trial in mainland China. 220 children aged 6-12 years with spherical equivalent range of -1.00 D to -6.00 D in both eyes were enrolled in Phase 1 for 1 year. Children who had completed the first year's follow-up continued in the second phase. In Phase 2, the placebo group was crossed over to the 0.01% atropine group (referred to as the 'placebo-atropine group'), and the 0.01% atropine group was crossed over to the placebo group (referred to as the 'atropine-placebo group'). All children underwent the examination of cycloplegic refraction and axial length at a 6-month interval. Only data from right eyes were included in analysis. Results One hundred thirty-three subjects completed 2 years of follow-up. In the first year, the mean myopia progression in atropine-placebo group was 0.21 +/- 0.08 D slower than that in placebo-atropine group. After cross-over treatment, the mean myopia progression in atropine-placebo group was 0.22 +/- 0.07D faster than that in placebo-atropine group in the second year. Over 2 years, the mean myopia progression was -1.26 +/- 0.66D and -1.25 +/- 0.70D in the atropine-placebo and placebo-atropine groups (p = 0.954). Conclusions The difference in myopia progression between atropine-placebo group and placebo-atropine group in Phase 1 was similar to Phase 2 during the cross-over treatment. Through our cross-over trial, the results suggest that there is no rebound effect after using 0.01% atropine eye drops to prevent progression of myopia.