Beta-hydroxyacyl-acyl carrier protein dehydratase (HAD) catalyzes the formation of enoyl-[ACP] from beta -hydroxylacyl-[ACP] and plays a key role in determining fatty acid composition during the process of oil biosynthesis. In this study, only one HAD gene (GenBank KU358903) was identified and isolated from tung tree (Vernicia fordii Hemsl.). The VfHAD contained an open reading frame (ORF) of 663 bp, encoding a polypeptide of 220 amino acid residues. It shared 86, 85, and 83% amino acid identities with the HADs of Hevea brasiliensis, Jatropha curcas, and Ricinus communis, respectively. The VfHAD exhibited all essential catalytic sites and the characteristic “hot-dog” structure of FabZ protein. Quantitative real-time PCR (qRT-PCR) analysis showed that VfHAD mRNA was detected in all tung tree organs, but its transcript was the most abundant in tung seeds at 20 weeks after flowering when oil rapidly accumulated. The VfHAD was localized in chloroplast. Heterologous expression of VfHAD significantly increased C16 fatty acids content in Escherichia coli, yeast, and tobacco. This study suggested that VfHAD may play a key role in fatty acid biosynthesis in tung seeds and could be a useful target for genetic improvement of oil quality.