We aimed to assess the effect of mannitol treatment on plant growth, as well as genomic DNA methylation levels and patterns in Arabidopsis thaliana by methylation-sensitive amplified polymorphism (MSAP) analysis. Mannitol at 50, 100, 150 and 200 mmol·L^–1 promoted the root length of Arabidopsis seedlings. MSAP analysis revealed that levels of 5-methyldeoxycytidine (5-mdC) in mannitol-treated seedlings were also altered significantly, from 17.75%, 21.15%, 15.49% to 46.10% in plants treated with 50, 100, 150 and 200 mmol·L^–1 mannitol, respectively. Thus, mannitol caused epigenetic changes in all samples based on different levels and patterns of DNA methylation. As the controls, methylation and demethylation of DNA in the seeds treated with mannitol was 5.78%, 15.48%, 10.71%, 33.73% and 10.98%, 5.36%, 8.33%, 7.69%, respectively. Mannitol causes a dose-dependent and transient change in global 5-mdC levels in Arabidopsis seedlings.