In bacteria, threonine dehydratases could convert L-threonine to 2-ketobutyrate. Some threonine dehydratases contain only a catalytic domain, while others contain an N-terminal catalytic domain and a C-terminal regulatory domain composed of one or two ACT-like subdomains. However, the role of the ACT-like subdomain in threonine dehydratases is not clear. Here, nine different bacterial threonine dehydratases were studied. Three of the nine contain no ACT-like subdomain, four of them contain a single ACT-like subdomain, and two of them contain two ACT-like subdomains. The nine genes encoding these threonine dehydratases were individually overexpressed in E. coli BL21(DE3), and the enzymes were purified to homogeneity. Activities of the purified enzymes were analyzed after incubation at different temperatures and different pHs. The results showed that threonine dehydratases with a single ACT-like subdomain are more stable at higher temperatures and a broad range of pH than those without ACT-like subdomain or with two ACT-like subdomains. Furthermore, the specific activity of threonine dehydratases increases with the increase of the number of ACT-like subdomains they contain. The results suggest that the ACT-like subdomain plays an important role in bacterial threonine dehydratases.