The influence of pesticide exposure in alteration of DNA methylation patterns of specific genes is still limited, specifically in natural antisense transcripts (NAT), such as the WRAP53α gene. The aim of this study was to determine the methylation of the WRAP53α gene in mestizo and indigenous populations as well as its relationship with internal (age, sex, and body mass index) and external factors (pesticide exposure and micronutrient intake). A cross-sectional study was conducted including 91 mestizo individuals without occupational exposure to pesticides, 164 mestizo urban sprayers and 189 indigenous persons without occupational exposure to pesticides. Acute pesticide exposure was evaluated by measurement of urinary dialkylphosphate (DAP) concentration by gas chromatograph coupled to a mass spectrometer. Anthropometric characteristics, unhealthy habits, and chronic pesticide exposure were assessed using a structured questionnaire. The frequency of macro- and micronutrient intake was determined using SNUT software. DNA methylation of the WRAP53α gene was determined by pyrosequencing of bisulfite-modified DNA. The mestizo sprayers group had the higher values of %5mC. In addition, this group had the most DAP urinary concentration with respect to the indigenous and reference groups. Bivariate analysis showed an association between %5mC of the WRAP53α gene with micronutrient intake and pesticide exposure in mestizo sprayers, whereas changes in %5mC of the WRAP53α gene was associated with body mass index in the indigenous group. These data suggest that the %5mC of the WRAP53α gene can be influenced by pesticide exposure and ethnicity in the study population, and changes in the WRAP53α gene might cause an important cell process disturbance.