Abstract Autophagy is required for the long-term maintenance of Ag-specific memory B cells. However, whether autophagy is also important for the initial formation of memory B cells remains unclear. In this study, we show that newly generated memory B cells do not display active autophagy but are capable of forming Ab-secreting cells after rechallenge with Ags. Increases in autophagy took place over time after the initial formation of memory B cells. The expression of transcription factors involved in autophagy, but not changes in epigenetic regulation by DNA methylation, was required for autophagy gene expression and the development of active autophagy in memory B cells. This indicates that autophagy is not critical for the initial generation of memory B cells but is required for their long-term persistence. Our results suggest that promoting autophagy to improve Ab-dependent immunological memory is more effective during memory B cell maintenance stage.