In order to effectively modulate the anabolism of carotenoid in tobacco, a β-carotene hydroxylase (BCH) gene related to plant carotenoid anabolism was cloned from Nicotiana tabacum. A cDNA sequence of encoded BCH gene was isolated from tobacco cv. K326 by using in silico cloning technique with pepper BCH gene sequence as a probe and tentatively designated as NtBCH1 (GenBank, JQ410446). NtBCH1 gene has a complete open reading frame encoding 309 amino acids with a conserved structural domain of FA_hydroxylase superfamily. The sequence comparison shows high homology to other plant BCH proteins of NtBCH1 protein, and the phylogenetic tree analysis indicates the closest genetic relationship between NtBCH1 and Solanum lycopersicum.