Background: Methamphetamine (METH) is one of the most widely distributed psychostimulants worldwide. Despite active counter measures taken by different countries, neither overall usage of METH nor the frequency of repeat users has reduced over the past decade. METH induces abuse and dependence as it acts on the central nervous system and temporarily stimulates the brain. The recidivism rate for abuse of stimulants in Japan is very high and therefore prevention of repeated usage is paramount. However, we lack information about the relationship between METH users and genomic changes in humans in Japan, which would provide important information to aid such efforts. Objective: Shati/Nat8l is a METH-inducible molecule and its overexpression has protective effects on the brain upon METH usage. Here we investigated the effect of METH usage on DNA methylation rates at the promoter site of SHATI/NAT8L. We used DNA samples from human METH users, who are usually difficult to recruit in Japan. Methods: We measured DNA methylation at SHATI/NAT8L promoter sites by pyrosequencing method using 193 samples of METH users and 60 samples of healthy subjects. In this method, DNA methylation is measured by utilizing the property that only non-methylated cytosine changes to urasil after bisulfite conversion. Results: We found that the rate of DNA methylation at six CpG islands of SHATI/NAT8L promoter sites is significantly higher in METH users when compared to healthy subjects. Conclusion: These results suggest that the DNA methylation rate of SHATI/NAT8L promotor regions offers a new diagnostic method for METH usage.