Several protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD(+) dependent formate dehydrogenase from Chaetomium thermophilum (CtFDH) can be utilized for interconversion of formate to carbon dioxide coupled with the conversion of NAD(+) to NADH. In this study, N-terminal His tagged CtFDH (N-CtFDH) and C-terminal His tagged CtFDH (C-CtFDH) was constructed to learn the effect of His tag location on the activity and kinetic parameters of the enzyme. The solubility of proteins was not affected by tag position, however, an interference on the N-terminal region caused a deterioration in specific activity and the kinetic ability of enzyme. The obtained results indicated that the C-terminus of the enzyme is an appropriate region for tag engineering. The C-CtFDH has an approximately three-fold larger specific activity and two-fold higher catalytic efficiency than N-CtFDH. The results suggest that insertion of a His-tag at the N-terminal or C-terminal end of CtFDH has different effects on the protein and the N-terminal fragment of the protein is crucial for the function of CtFDH.