Thermoelectric performance of Ca3Co4O9 system has been enhanced through the misplaced substitution with Cr doping at Ca-sites. The study of Cr doping at Co-sites has also been performed for comparing. Base on the analysis of the structural data, we conclude that the doped Cr ions should be in the form of Cr+6, and enter into Ca-sites of Ca2CoO3 layers in Ca3-xCrxCo4O9 lattice and Co-sites of CoO2 layers in Ca3Co3.90Cr0.10O9 lattice, respectively. As Cr ions are doped into Ca-sites in Ca3-xCrxCo4O9 lattice, both resistivity and thermopower increase obviously, while thermal conductivity decreases. Among all samples, Ca2.95Cr0.05Co4O9 shows the maximum ZT value, which is enhanced by 12% than that of Ca3Co4O9. The results show that the misplaced substitution with a proper Cr doping at Ca-sites is beneficial to the enhancement of the thermoelectric performance in Ca3Co4O9 system compared to the traditional idea: Cr doping at Co-sites, which is suggested to originate from the combined action of the carrier concentration, the electronic correlation, and the lattice disharmony.