Thiamine deficiency (TD) impairs hippocampal neurogenesis. However, the mechanisms involved are not identified. In this work, TD mouse model was generated using a thiamine-depleted diet at two time points, TD9 and TD14 for 9 and 14 days of TD respectively. The activities of pyruvate dehydrogenase (PDH), alpha-ketoglutamate dehydrogenase (KGDH), glucose-6-phosphate dehydrogenase (G6PD), and transketolase (TK), as well as on the contents of NADP(+) and NADPH were determined in whole mouse brain, isolated cortex, and hippocampus of TD mice model. The effects of TK silencing on the growth and migratory ability of cultured hippocampal progenitor cells (HPC), as well as on neuritogenesis of hippocampal neurons were explored. The results showed that TD specifically reduced TK activity in both cortex and hippocampus, without significantly affecting the activities of PDH, KGDH, and G6PD in TD9 and TD14 groups. The level of whole brain and hippocampal NADPH in TD14 group were significantly lower than that of control group. TK silencing significantly inhibited the proliferation, growth, and migratory abilities of cultured HPC, without affecting neuritogenesis of cultured hippocampal neurons. Taken together, these results demonstrate that decreased TK activity leads to pentose-phosphate pathway dysfunction and contributes to impaired hippocampal neurogenesis induced by TD. TK and pentose-phosphate pathway may be considered new targets to investigate hippocampal neurogenesis.