Thermoelectric materials, which allow the conversion between heat and electricity, can be directly applied in the fields of cooling and power generation. Here we report an effective approach: non-isoelectronic substitution of Sb for Cu and Te in Cu3Ga5Te9 to increase the Seebeck coefficient and electrical conductivity. This improvement is attributed to the enhancement in carrier concentration n and effective mass as well as the conservation of the carrier mobility μ. The enhancement of the carrier concentration is caused by the hole doping effect due to the drop of the Fermi level into the valence band when Sb occupies the Te lattice sites, and also due to the increase of the copper vacancy (V-1Cu) concentration when Cu content decreases. In addition, the non-isoelectronic substitution can yield extra crystal structure defects. These defects, which are represented by the alterations of anion (Te2-) position displacement (u) and tetragonal deformation (η), directly govern the lattice thermal conductivity (κL) on an atomic scale. The maximum ZT value is 0.6 at 766 K with proper Sb substitution, which is about 25% higher than that of Cu3Ga5Te9. Therefore, we are able to effectively manipulate the electrical and thermal properties through proper selections of the substituting / substituted elements and their quantities, and prove that the non-isoelectronic substitution approach in the chalcopyrite semiconductors is an effective way to improve the thermoelectric performance.