In the current study, a three-tiered mutagenesis strategy was employed to simultaneously improve the thermostability and activity of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) by engineering the last ten amino acids (Met245∼Glu254) of its C-terminal region. Initially, truncated mutagenesis results displayed that C-terminal deletions decreased the thermostability and/or activity of HheC. Then ten residues were subjected to single-site saturation mutagenesis, resulting in 20 beneficial single-point variants related to the thermostability or activity of HheC. The results clearly indicated that residues Met252∼Glu254 and Trp249 are crucial for regulating enzyme thermostability and activity, respectively. Finally, the beneficial substitutions were combined using efficient multi-site combinatorial mutagenesis approaches, leading to an outstanding variant PX14 (Trp249Pro/Met252Leu/Pro253Asp), which had a 17.8-fold higher half-life and a 4.0-fold higher kcat value than that of wild-type HheC. These results indicated that the C-terminal residues play an important role in modulating both the thermostability and activity of HheC.