Exogenous DNA localization and the frequency of spermatozoa carrying exogenous DNA after sperm/DNA co-culture are key to a successful sperm mediated-gene transfer (SMGT). In the study, the characteristics and influencing factors of exogenous DNA uptake by spermatozoa were tested using digoxigenin (DIG) labeled DNA as trace. Results showed that goat spermatozoa could spontaneously take up exogenous DNA. The exogenous DNA was initially bound to the outer sperm membrane at postacrosomal region; subsequently party of the bound DNA was internalized into nucleus. There were considerable differences in the capability of spermatozoa from different donors to bind and internalize exogenous DNA. In 35 samples, binding rates (before DNase I digestion) and internalization rates (the positive rate after DNase I digestion) varied between 4.6%-62.4% and 2.1%-53.8%, respectively. For the spermatozoa from the same goat, the binding and internalization capacities were mostly inhibited by the seminal fluid. Compared to ejaculate sperm, the binding rate and internalization rate were increased three and five times in washed sperm cells, respectively. At the same time, capacitated spermatozoa also had lower exogenous DNA uptake (P<0.01). Dead spermatozoa did not complete the internalization process. The highest positive rate (before DNase I digestion) was found in membrane-broken spermatozoa as a result of freeze-thawing and this was independent of the sperm donors. These results suggest that selection of appropriate sperm donors and optimization of sperm processing procedures are the key steps for successful SMGT.