SUMMARYThiamine functions as a crucial activator modulating plant health and broad‐spectrum stress tolerances. However, the role of thiamine in regulating plant virus infection is largely unknown. Here, we report that the multifunctional 17K protein encoded by barley yellow dwarf virus‐GAV (BYDV‐GAV) interacted with barley pyrimidine synthase (HvTHIC), a key enzyme in thiamine biosynthesis. HvTHIC was found to be localized in chloroplast via an N‐terminal 74‐amino acid domain. However, the 17K‐HvTHIC interaction restricted HvTHIC targeting to chloroplasts and triggered autophagy‐mediated HvTHIC degradation. Upon BYDV‐GAV infection, the expression of the HvTHIC gene was significantly induced, and this was accompanied by accumulation of thiamine and salicylic acid. Silencing of HvTHIC expression promoted BYDV‐GAV accumulation. Transcriptomic analysis of HvTHIC silenced and non‐silenced barley plants showed that the differentially expressed genes were mainly involved in plant–pathogen interaction, plant hormone signal induction, phenylpropanoid biosynthesis, starch and sucrose metabolism, photosynthesis‐antenna protein, and MAPK signaling pathway. Thiamine treatment enhanced barley resistance to BYDV‐GAV. Taken together, our findings reveal a molecular mechanism underlying how BYDV impedes thiamine biosynthesis to uphold viral infection in plants.