Behavioral flexibility is in close proximity to dentate gyrus (DG) function and long-term depression (LTD), but the role of DG LTD in behavioral flexibility has hitherto been unexplored. Although the functions of alpha-Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been studied extensively, the role of βCaMKII, a constituent of the CaMKII holoenzyme, in LTD and behavioral flexibility has not been investigated in vivo. Here using the βCaMKII-F90G transgenic (TG) mice, in which the inducible and reversible overexpression of βCaMKII is restricted to dentate gyrus (DG), we found that TG mice exhibited defective behavioral flexibility in two reversal tasks and seriously impaired N-methyl-D-aspartic acid receptor (NMDAR)-dependent LTD in DG medial perforant path (MPP). Consistent with the deficit in NMDAR-LTD, GluA1-Ser845, GluA1-Ser831 dephosphorylation and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalization were also disrupted during NMDAR-LTD in TG mice. Furthermore, these deficits were due to decreased activities of protein phosphatases (PP) 1/2A and glycogen synthesis kinase 3 beta (GSK3β), and overexpressed synaptic stargazin in TG mice. Importantly, all the deficits above could be reversed by 1-naphthylmethyl (NM)-PP1, a specific inhibitor of the exogenous βCaMKII-F90G. Taken together, our findings for the first time demonstrate that βCaMKII overexpression impairs behavioral flexibility and NMDAR-dependent LTD in DG MPP, which further confirms the close relationship between NMDAR-dependent LTD and behavioral flexibility.