N-ethyl-N-nitrosourea (ENU) is a potent mutagen in a mouse model by inducing point mutation in a random manner and, in particular, causing heritable base substitutions in spermatogonia. In this study, systematic development of phenotype-driven mutant mice with large scale was carried out by using ENU. Nine-week-old male mice of C57BL/6J received intraperitoneal injection at three times with 100 mg/kg of ENU at weekly intervals for three weeks. After injections with ENU, the changes of body weight, fatality, recovery of fertile period, and breeding record were measured in these mice. Body weight lost as a result of ENU treatments was reversed after the last ENU injection. Live fertile male mice recovered from infertility from 104 to 165 days after ENU treatments were mated with C57BL/6J female mice for generation of G1 offspring. An average birth rate was 5.9 mice from 1 pair of paternal and maternal mice. All of 231 G1 offspring mice were analyzed by modified-SHIRPA with standard procedure at nine weeks of age. Among G1 mice, 166 mice were identified as mutagenic phenotypes in 20 test items. The changes in mutagenic phenotypes after ENU treatments, for instance, pattern in the region with a different color, touch escape, changes in head morphology, pupil, and teeth, and negative geotaxis etc., were found in these mice. Taken together, these results indicate that ENU may be a trans-generational mutagen in C57BL/6J mice.