Sperm-mediated gene transfer (SMGT) can be used to transfer exogenous DNA into the oocyte at fertilization. The main objective of this study was to assess efficiency of transferring mitochondrial DNA (mtDNA) fragment into boar spermatozoa in either presence or absence of liposome and quality of transfected spermatozoa. The mtDNA of chicken liver was isolated and purified by phenol and alkaline lysis extraction, and it was inserted to plasmid. The genome of transfected spermatozoa treated with DNase Ⅰ was purified by alkaline lysis, and then amplified by the PCR analysis. After electrophoresis, DNA quantitation of each well was calculated by comparison of the band intensity with standard. As a result, exogenous DNA was composed of mtDNA fragment (1.2 kb) and plasmid (2.7 kb). On the other hand, efficiency of transfection by liposome (9.0±0.34 ng/l) in SMGT was higher than that by DNA solution (6.9±0.53 ng/l). However, there was no significant difference. Transfering exogenous DNA into spermatozoa was completed within 90 min of incubation. In another experiment, there were significant (p<0.05) differences between transfected spermatozoa using both DNA solution and DNA/liposome complexes with untreated spermatozoa for viability (70.8±1.80 and 68.0±2.16% vs. 83.3±1.69%, respectively) and motility (78.7±1.59 and 79.3±2.14% vs. 86.7±1.59%, respectively). This study indicates that exogenous mtDNA can be efficiently transferred into boar spermatozoa regardless of the presence of liposome, and transfected spermatozoa can also use insemination and in vitro fertilization to generate transgenic pig.