The present study is expected to provide better understanding of how exo-β-glucanase acts on the biochemo-rheological property of the cell wall, and thus the regulation mechanism of pollen tube growth. A polymerase chain reaction (PCR)-based strategy and cDNA library screening were used to clone an exo-β-glucanase gene (LP-ExoI) from Lilium longiflorum pollen tubes. The LP-ExoI sequence showed a high level of homology to that of reported plant exo-β-glucan hydrolases. Phylogenetic analysis demonstrated that there was close relationship between LP-ExoI and other exo-glucan hydrolases in Poacea plants. Northern analysis indicated that LP-ExoI transcripts stored in pollen grains at low levels; LP-ExoI transcription level enhanced at 2 h after the onset of pollen incubation and remained relatively high during pollen tube elongation. Moreover, the expression of LP-ExoI was undetectable in petals, filaments, stigmas, styles, ovaries, leaves, and stems. These results suggest that LP-ExoI gene is a late pollen gene, specifically contributing to the regulation of lily pollen germination and pollen tube growth.