LINE-1 elements comprise approximately 17% of human DNA and their mobility continues to impact genome evolution. However, little is known about the types of non-transformed cells that can support LINE-1 retrotransposition. Here, we show that human embryonic stem cells express endogenous LINE-1 elements and can accommodate LINE-1 retrotransposition in vitro. The resultant retrotransposition events can occur into genes and can result in the concomitant deletion of genomic DNA at the target site. Thus, these data suggest that LINE-1 retrotransposition events may occur during early stages of human development.