EMS mutant of NtHMA4，hma44h12，was screened from mutant library of main tobacco cultivar ‘Yunyan 87’ in China by TILLING technology in this study. Sequencing of the mutants showed that the mutation of nucleotides was C152T in the hma44h12, and the mutation of amino acid in the hma44h12 was T51I. The content of cadmium, zinc, copper, arsenic, lead and nickel in hma44h12 leaf were decreased by 55.48%, 21.11%, 20.96%, 27.50%, 16.23%, and 19.28% respectively compared with those of control after 100 μmol/L CdCl₂ treatment. hma44h12 was planted in the field and its agronomic traits were observed. Plant height, stem circumference and intermodal length of hma44h12 were decreased significantly compared with those of control. It was speculated that hma44h12 contained mutant loci affecting these traits. The low cadmium mutant, hma44h12, obtained from this study can be used as a material for cultivating low cadmium tobacco varieties. However, the use of this mutant in breeding requires further backcrossing to remove the negative mutation.