Cordyceps militaris has been used in traditional Chinese medicine for many years, but its frequent degeneration during continuous maintenance in culture can lead to substantial commercial losses. In this study, a degenerated strain of C. militaris was obtained by subculturing a wild-type strain through 10 successive subcultures. The relative abundance of the 2 mating types seems to be out of balance in the degenerated strain. By cross-mating 4 single-ascospore isolates (2 for MAT 1-1 and 2 for MAT 1-2) from the degenerated strain, we were able to restore fruiting body production to wild-type levels. The rejuvenated strain not only produced well-developed fruiting bodies but also accumulated more cordycepin and adenosine than either the original wild-type strain or the degenerated strain. These new characteristics remained stable after 4 successive transfers, which indicates that the method used to rejuvenate the degenerated strain in this study is an effective approach.