Transgenic (Tg) animals are widely used in researching the characteristics of exogenous genes. Intracytoplasmic sperm injection (ICSI)-mediated transgenesis (ICSI-Tr) has been a useful method for generating Tg animals, especially in the mouse. However, the original methods using freeze-thawed spermatozoa showed severe chromosomal damage and low offspring rates after embryo transfer. Herein, we describe an improved method to generate Tg mice efficiently using a simple pretreatment of spermatozoa with 10 mM NaOH. These spermatozoa lost their plasma membrane and tail, while still maintaining nuclear integrity. Sperm heads were mixed with 0.5–5 ng/μl of the transgene for enhanced green fluorescent protein (EGFP) for 3 min to 1 h at room temperature and were then microinjected into oocytes by ICSI. The best results were obtained when treated spermatozoa were incubated with 2 ng/μl of EGFP for 10 min; 55.6% of injected embryos developed to the blastocyst stage, and more than half (56.9%) of them displayed EGFP fluorescence. Under these conditions, 12 pups of 34 offspring were positive for the transgene after transfer at the 2-cell stage into pseudopregnant recipient mice (a high rate [10.2%] from manipulated embryos). This method was found to be suitable for hybrid and inbred strains of mouse such as C57BL/6 and 129X1/Sv. Thus, a simple sperm pretreatment with NaOH before ICSI-Tr resulted in an efficient insertion of an exogenous gene into the host genome. This method allows for easy production of Tg mice, requiring fewer oocytes for micromanipulation than classical methods.