SUMMARY The THIAMIN REQUIRING2 (TH2) protein comprising a mitochondrial targeting peptide followed by a transcription enhancement A and a haloacid dehalogenase domain is a thiamin monophosphate (TMP) phosphatase in the vitamin B1 biosynthetic pathway. The Arabidopsis th2‐3 T‐DNA insertion mutant was chlorotic and deficient in thiamin diphosphate (TDP). Complementation assays confirmed that haloacid dehalogenase domain alone was sufficient to rescue the th2‐3 mutant. In pTH2:TH2‐GFP/th2‐3 complemented plants, the TH2‐GFP was localized to the cytosol, mitochondrion, and nucleus, indicating that the vitamin B1 biosynthetic pathway extended across multi‐subcellular compartments. Engineered TH2‐GFP localized to the cytosol, mitochondrion, nucleus, and chloroplast, could complement the th2 mutant. Together, these results highlight the importance of intracellular TMP and thiamin trafficking in vitamin B1 biosynthesis. In an attempt to enhance the production of thiamin, we created various constructs to overexpress TH2‐GFP in the cytosol, mitochondrion, chloroplast, and nucleus. Unexpectedly, overexpressing TH2‐GFP resulted in an increase rather than a decrease in TMP. While studies on th2 mutants support TH2 as a TMP phosphatase, analyses of TH2‐GFP overexpression lines implicating TH2 may also function as a TDP phosphatase in planta. We propose a working model that the TMP/TDP phosphatase activity of TH2 connects TMP, thiamin, and TDP into a metabolic cycle. The TMP phosphatase activity of TH2 is required for TDP biosynthesis, and the TDP phosphatase activity of TH2 may modulate TDP homeostasis in Arabidopsis.