Phaeodactylum tricornutum is a model microalgae that is widely used to study diatom physiology and ecology. Since the meiotic process and sexual cycle have never been observed directly, P. tricornutum has been considered to be an asexual species. However, phylogenetic analysis of the P. tricornutum genome has revealed a series of meiosis-specific gene homologues in this species. We identified two copies of differently transcribed SPO11 homologs that contain the conserved motifs of Winged-helix and Toprim domains. The homolog PtSPO11-3 interacts with TopoVIB in yeast two-hybrid analysis, whereas the homolog PtSPO11-2 could rescue the sporulation defect of a Spo11 yeast mutant strain. PtSPO11-2 was also found to be significantly up-regulated at low temperatures in P. tricornutum and its key catalytic residue was important to the homolog's function in sporulation. The results herein provide positive clue that meiosis and sexual reproduction could exist in this diatom.