Ser‐Ala and Ser‐Ala‐Ser‐Ala C‐terminus elongated (Δ+2 and Δ+4, respectively) and two C‐terminus deleted (Δ−2 and Δ−3) muscle acylphosphatase mutants were investigated to assess the catalytic and structural roles of the C‐terminal region. The kinetic analysis of these mutants shows that the removal of two or three C‐terminal residues reduces the catalytic activity to 7% and 4% of the value measured for the wild‐type enzyme, respectively; instead, the elongation of the C‐terminus does not significantly change the enzyme behaviour.1H Nuclear magnetic resonance spectroscopy indicates that all mutants display a native‐like fold though they appear less stable, particularly Δ−2 and Δ−3 mutants, as compared to the wild‐type enzyme. Such destabilisation of the C‐terminal modified mutants is further confirmed by urea inactivation experiments. The results here presented account for an involvement of the C‐terminal region in the stabilisation of the three‐dimensional structure of acylphosphatase, particularly at the active‐site level. Moreover, a participation of the C‐terminal carboxyl group to the catalytic mechanism can be excluded.