Although injected freeze-dried hamster spermatozoa can develop into male pronuclei even after 12 months of storage at 4°C, the developmental competence of hamster pronuclear oocytes is not well understood. Furthermore, production of live offspring from freeze-dried spermatozoa is limited in some animals, such as mice, rabbits and rats. Here, we report the birth of hamster offspring following intracytoplasmic injection with freeze-dried spermatozoa. The integrity of the sperm DNA after freeze-drying and rehydration is very important for the developmental competence of hamster embryos produced by injecting freeze-dried spermatozoa into oocytes. This study used the TUNEL method to examine DNA fragmentation and the chromosomal integrity of spermatozoa after freeze-drying using two freezing media: M2 medium, and a Tris-HCl buffered solution containing 50 mM EGTA (EGTA solution). The rate of DNA fragmentation when hamster spermatozoa freeze-dried in EGTA solution was used was significantly (P < 0.05) lower than that in M2 medium (4.3% vs. 41.4%), and the chromosomal integrity in EGTA solution was higher in EGTA solution than in M2 medium (81.1% vs. 41.0%). The percentage of morulae/blastocysts derived from hamster spermatozoa freeze-dried in EGTA solution was significantly (P < 0.05) higher than that derived from spermatozoa freeze-dried in M2 medium (62.2% vs. 12.5%). After transfer to foster mothers, 3 of 23 morulae/blastocysts developed into live offspring.