All living organisms face a challenge to sense and respond appropriately to the environmental cues. In C. elegans, the FoxO transcription factor DAF-16 translocates into nucleus upon stress to regulate gene expression for organismal survival. The subcellular localization and activity of DAF-16 are tightly controlled by its post-translational modifications, including phosphorylation, acetylation, ubiquitination, methylation and glycosylation. In our previous study, we found that sirtuin family protein SIR-2.4, a homolog of mammalian SIRT6 and SIRT7, promotes DAF-16 nuclear translocation and DAF-16-dependent transcription under stress conditions. SIR-2.4 acts antagonistically with the acetyltransferase CBP-1 to negatively regulate DAF-16 acetylation. However, this modulation does not require the catalytic activity of SIR-2.4. We further identified four CBP-1-mediated acetylation sites on DAF-16 by mass spectrometry analysis. Mutations on some of these acetylation sites alter the kinetics of DAF-16 translocation upon stress, indicating a new nuclear localization signal of DAF-16. Furthermore, mutations on these acetylation sites affect both the lifespan and stress resistance of the animals, possibly due to an increased DAF-16 transactivating activity. Together, our studies identify acetylations on DAF-16 protein that significantly affect its functions in response to stress.