The honeybee is a model organism for studying learning and memory formation and its underlying molecular mechanisms. While DNA methylation is well studied in caste differentiation, its role in learning and memory is not clear in honeybees. Here, we analyzed genome-wide DNA methylation changes during olfactory learning and memory process in A. mellifera using whole genome bisulfite sequencing (WGBS) method. A total of 853 significantly differentially methylated regions (DMRs) and 963 differentially methylated genes (DMGs) were identified. We discovered that 440 DMRs of 648 genes were hypermethylated and 274 DMRs of 336 genes were hypomethylated in trained group compared to untrained group. Of these DMGs, many are critical genes involved in learning and memory, such as Creb, GABA B R and Ip3k, indicating extensive involvement of DNA methylation in honeybee olfactory learning and memory process. Furthermore, key enzymes for histone methylation, RNA editing and miRNA processing also showed methylation changes during this process, implying that DNA methylation can affect learning and memory of honeybees by regulating other epigenetic modification processes.