The basic helix-loop-helix (bHLH) protein Mist1 is an important exocrine pancreas transcriptional factor expressed in the acinar cells of mammals. In the present study, we cloned the homologous Mist1 cDNA encoding a predicted protein of 184 amino acids in zebrafish. The typical bHLH domain of zebrafish Mist1 shares high identity with that of its orthologs in mouse, rat, and human. Expression analysis revealed that Mist1 maternal transcripts are distinct in the very beginning of embryogenesis and that endogenous Mist1 is chronologically expressed in polster, hatching gland, hindbrain and appears exclusively in the pancreas from 72 hpf onward. Knockdown of Mist1 conditionally causes mild morphological defects in embryos. In MO-treated embryos, midbrain-hindbrain boundary is missing and exocrine pancreas is significantly reduced and disorganized. These results suggest that Mist1 functions in an evolutionary conserved way as a key transcriptional regulator specific for exocrine pancreas development in zebrafish.