Vitamin B1 (VB1) plays a crucial role in sustaining plant health and enabling adaptive responses to environmental stress. The complex maize genome implies a sophisticated VB1 synthesis pathway, with the mechanisms by which VB1 benefits plants remaining elusive. Here, we identified two VB1 biosynthetic genes, THIAMINE REQUIRING 1 (ZmTH1) and its paralog THIAMINE MONOPHOSPHATE SYNTHASE 1 (ZmTMPS1), from a natural mutant pale leaf and depauperate growth 1 (pldg1). We elucidated their specific roles in regulating multiple thiamin diphosphate (TDP)-dependent metabolic pathways and their effects on plant growth and stress tolerance. ZmTH1 encodes a chloroplast-localised,bifunctional enzyme comprising phosphomethylpyrimidine kinase (HMPP-K) and thiamine monophosphate synthase (TMP-S) domains. Functional dissection revealed that these domains functioned synergistically,with disruption of one domain significantly attenuating the other,although both can function independently. A frameshift mutation in ZmTH1 (Zmth1) resulted in reduced biosynthesis of VB1,TMP and TDP. Consequently,the activity of TDP-dependent enzymes was impaired,disrupting multiple TDP-dependent metabolic pathways. Additionally, ZmTMPS1, localised to the cytosol and nucleus, exhibited limited TMP-S activity that partially compensated for ZmTH1 mutation in pldg1 but cannot fully restore VB1 levels. Overexpression of ZmTH1 or exogenous VB1 application enhanced maize seedling tolerance to cold and drought stresses by increasing TDP-dependent enzyme activity. These findings advance the understanding of VB1 metabolism in maize and provide genetic targets for improving stress resilience and crop performance.