As self-subunit swapping chaperones or metallochaperones, the activators assist nitrile hydratases to take up metal ions and they are essential for active expression of nitrile hydratases. Compared with nitrile hydratases, the activators have a low sequence identity. Study of the activation characteristics and the relationships between structures and functions of the activators is of great significance for understanding the maturation mechanism of nitrile hydratase. We co-expressed low-molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1 with four heterologous activators respectively and determined their activation abilities. Then we made sequence analysis and structure modelling, and studied the functions of the important domains of the activators. Results showed that all four heterologous activators could activate L-NHase, however, the specific activities of L-NHases were different after activation. L-NHase showed the highest specific activity after being activated by activator A, which was 97.79% of that of the original enzyme, but the specific activity of L-NHase after being activated by activator G was only 23.94% of that of the original enzyme. Activator E and activator G had conserved domains (TIGR03889), and deletion of their partial sequences resulted in a substantial loss of activation abilities for both activators. Replacing the N-terminal sequence of activator G with the N-terminal sequence of activator E, and adding the C-terminal sequence of activator E to the C-terminus of activator G could increase the specific activity of L-NHase by 178.40%. The activation by nitrile hydratase activators was universal and specific, and the conserved domains of activators were critical for activation, while the N-terminal domain and C-terminal domain also had important effects on activation.