Cyclin-dependent kinase 5(Cdk-5) and protein kinase A(PKA) are the important kinases in the regulation of Tau phosphorylation. However, it is unclear weather they participate in the regulation of tau phosphorylation in diabetic rat brain. The roles of Cdk-5 and PKA in the regulation of abnormal tau hyperphosphorylation in the hippocampus of diabetic rat model have been investigated. The diabetic rat model was induced by intra peritoneal injection with 55 mg/kg streptozotocin. Intracellular free calcium concentration was detected by loading Fura-2 and fluorescent technique in Control, Con+ Ros, DM, and DM+Ros groups, and the activities of Cdk-5 and PKA were measured by immunoprecipitation and by liquid scintillation for incorporated radioactivity respectively, furthermore, Western blotting was used to examine the level of tau phosphorylation by using phosphorylation-dependent and site-specific tau antibodies. The results show that, compared to normal control, the concentration of intracellular calcium in the diabetic rat hippocampus was elevated to 165.92 nmol/L(P < 0.05). Cdk-5 activity was increased by 29% of control, and an increase of PKA activity by 30% of control was also found, both Cdk-5 activity and PKA activity were increased highly compared to normal control(P < 0.01, P < 0.01), furthermore, the immunoreactivity of Tau-1(detection of Ser 198/Ser 199/ Ser202, non-phosphorylated site) was decreased(P < 0.01), the immunoreactivity of PHF-1 sites (detection of ser396/ser404, phosphorylated site) was increased(P < 0.05) in these diabetic rats. After treated the DM rats with roscovitine, a specific Cdk-5 inhibitor, Cdk-5 activity was inhibited to 105% vs control, it was not obviously increased compared to control (P >0.05), but PKA activity remained 127% vs control, it was high compared to control(P < 0.01). Moreover, the hyperphosphorylation of tau at Ser198/Ser199/Ser202 epitopes was reversed (P < 0.01), but the level of phosphorylation of tau at Ser396/Ser404 epitopes was remained highly after roscovitine was administered to DM rats. However, after treated the control rats with roscovitine, Cdk-5 activity was about 104% vs control, and PKA activity was 105% vs control, neither Cdk-5 activity nor PKA activity was higher compared to control(P > 0.05), and the level of tau phosphorylation at Ser198/Ser199/Ser202 and Ser396/Ser404 sites was not increased compared to control too(P > 0.05). These results firstly suggest that the increase of Cdk-5 activity and PKA activity may cause the hyperphosphorylation of tau at Ser198/Ser199/Ser202 and Ser396/Ser404 epitopes in diabetic rat hippocampus, and the intracellular calcium may play a role in this process, however, it is needed to be elucidated in great details.