Spaceflight represents a unique environmental condition whereby dysregulated gene expression and genomic instability can be provoked. However, detailed molecular characterization of the nature of genetic changes induced by spaceflight is yet to be documented in a higher eukaryote. Transposable elements (TEs) are ubiquitous and have played a significant role in genome evolution. Mounting evidence indicates that TEs constitute the genomic fraction that is susceptible and responsive to environmental perturbations, and hence, most likely manifesting genetic instabilities in times of stress. A predominant means for TEs to cause genetic instability is via their transpositional activation. Here we show that spaceflight has induced transposition of several endogenous TEs in rice, which belong to distinct classes including the miniature inverted terminal repeat TEs (MITEs) and long-terminal repeat (LTR) retrotransposons. Of three rice lines studied, transposition of TEs were detected in the plants germinated from space-flown dry seeds of two lines (RZ1 and RZ35), which are genetically homogeneous and stabilized recombinant inbred lines (RILs) derived from a pure-line rice cultivar, Matsumae. In contrast, the TEs remained immobile in plants derived from space-flown seeds of Matsumae itself, indicating a genotype-dependent manner of TE transposition under the spaceflight environment. Further examination showed that at least in some cases transposition of TEs was associated with cytosine demethylation within the elements. Moreover, the spaceflight-induced TE activity was heritable to organismal progenies. Thus, our results implicate that the spaceflight environment represents a potent mutagenic environment that can cause genetic instabilities by eliciting transposition of otherwise totally quiescent endogenous TEs in a higher eukaryote.