Preservation of mammalian spermatozoa now plays an important role in fertility treatment, in generating hybrid animals, and in protecting endangered or extinct species. To date, the most common method of sperm preservation is freezing in liquid nitrogen (LN₂). However, this method requires constant supplementation of the LN₂ and also involves some safety issues in transporting LN₂. Here we describe a new sperm preservation method that does not involve freezing. Mouse spermatozoa were cultured in four basic media (HEPES-CZB, potassium simplex optimization medium with amino acids [KSOMaa], K -rich nuclear isolation medium [NIM], and PBS) with or without 10% bovine serum albumin (BSA) or 15% Ficoll as a protectant, and preserved in a refrigerator for up to 6 mo. These preserved sperm were then injected into fresh oocytes and cultured to the blastocyst stage in vitro or transferred into recipient females to demonstrate their genetic integrity. The results of sperm preservation for 1 mo suggested that NIM and PBS were better media than HEPES-CZB or KSOMaa and that BSA and Ficoll could improve either blastocyst or full-term development. Surprisingly, 18 pups were obtained using spermatozoa stored in these media for 6 mo. Moreover, this new method allowed easy production of healthy offspring even after transport of spermatozoa between two countries by aircraft at room temperature. In conclusion, this method allows for easy long-term preservation of mouse spermatozoa in a simple, modified medium at refrigerator temperature with very low cost and wide application.