Spermiogenesis is the step during which post-meiotic cells, called spermatids, undergo numerous morphological changes and differentiate into spermatozoa. Thousands of genes have been described to be expressed at this stage and could contribute to spermatid differentiation. Genetically-engineered mouse models using Cre/LoxPor CrispR/Cas9 are the favored approaches to characterize gene function and better understand the genetic basis of male infertility. In the present study, we produced a new spermatid-specificCretransgenic mouse line, in which the improvediCrerecombinase is expressed under the control of theacrosomal vesicle protein 1gene promoter (Acrv1-iCre). We show that Cre protein expression is restricted to the testis and only detected in round spermatids of stage V to VIII seminiferous tubules. TheAcrv1-iCreline can conditionally knockout a gene during spermiogenesis with a > 95% efficiency. Therefore, it could be useful to unravel the function of genes during the late stage of spermatogenesis, but it can also be used to produce an embryo with a paternally deleted allele without causing early spermatogenesis defects.