AMP-activated protein kinase (AMPK) functions as a cellular energy sensor by monitoring the cellular AMP:ATP ratio and plays a central role in cellular and whole-body energy homeostasis. Recent studies have suggested that AMPK also contribute to cell cycle regulation, but its role in this field remains almost elusive. In the present study, we report that AMPK alpha 1 was transiently activated during G(1)/S transition phase in NIH3T3 cells in the absence of any metabolic stress. Inhibition of AMPK activity at G(1)/S transition phase completely blocked cells from entering S phase; in contrast, persistent activation of AMPK at G(1)/S transition phase allowed cells to normally enter S phase, but these cells failed to proceed to G(2)/M phase, stacking at S phase. We further demonstrated that activation of AMPK at G(1)/S transition phase depends on Ca2+ transients and caMKK beta activity, but not on energy status. Collectively, these data indicate that temporal regulation of AMPK is required for proper control of S phase in NIH3T3 cells. (C) 2018 Elsevier Inc. All rights reserved.