Optimization of in vitro methods for the production of maize double haploids plays an important role in the breeding programs of this plant. In this study, the effects of 5-azacytidine on agronomic traits, androgenesis induction efficiency and also, DNA methyltransferase gene expression (AF229183.1) in two growth stages of maize were investigated. This experiment was performed as factorial based on a completely randomized block design with three replications. Two maize genotypes (DH5 × DH7 and ETMH-82) were considered as the first factor and treatment of maize seeds with 5-azacytidine (0, 5, 10, and 100 μM) was considered as the second factor. The maize seeds were sowed in the field and during the growth stages, various morphological and agronomic traits were recorded. In the anther culture experiment, the suitable anthers containing microspores at mid to late-uninucleate stages were selected and cultured in an YPm culture medium containing 1 mg/l 2, 4-D, and 2 mg/l BAP. Interaction effects of genotype and 5-azacytidine concentrations showed significant differences for the majority of studied traits except for number of kernel per ear row, kernel depth, plant diameter, number of leaves and number of ears. The highest amounts of 1000-kernel weight were obtained with treatments of 10 and 100 μM and the highest ones for grain yield and biological yield traits were obtained with 100 μM 5-azacytidine treatment for both genotypes. Seeds of DH5 × DH7 genotype treated with 5 μM 5-azacYtidine produced the highest mean number of embryo-like structures (0.1833) and regenerated plantlets (0.067) per each anther. Relative expression of DNA methyltransferase gene in maize seeds treated with different concentrations of 5-azacytidine showed a significant decrease in both genotypes and both growth stages compared to control plants (treated with 0 μM 5-azacytidine), that this decrease in gene expression could lead to improved androgenesis induction in anther culture of DH5 × DH7 genotype. However, despite the decrease in expression of this gene in two growth stages of ETMH-82 genotype, androgenesis induction was not observed in this genotype. The results of the present study can help to determine the role of epigenetic factors in androgenesis induction and improving the production of haploid plants in maize.