Long-term memory (LTM) in Drosophila is formed after spaced training, multiple trainings with inter-trial intervals (ITIs) between each training trial. LTM is not formed after massed training, multiple trainings without ITIs. A previous report demonstrates that MAP kinase (MAPK) activity increases specifically during the ITIs and decreases during trainings, leading to phasic MAPK activity. However, it has not been clear exactly what function of ITI dependent MAPK activity during spaced training. In this study, we focused on two transcription factors, CREB and c-fos. Both transcription factors are phosphorylated by MAPK and we found that expression of both CREB activator and c-fos increase during spaced training. Expression of either a dominant negative form of c-fos, or a CREB repressor, prevented training-dependent increases in both transcription factors. This indicates that LTM-dependent expression of CREB activator requires c-fos, while LTM-dependent expression of c-fos requires CREB activator. To determine where training-dependent increases in CREB and c-fos occur in the brain, we performed immunostaining experiments and found that increases in pMAPK, CREB, and c-fos activity occur in overlapping cells. Our results suggest that c-fos and CREB form a positive feedback transcriptional loop during spaced training that is required for LTM formation.