Actin depolymerizing factor (ADF) plays an important role in regulating actin assembly and disassembly for cells. In this study, the genomic sequence of HbADF, a 2 258 bp-length with two introns and three exons, was obtained from Hevea brasiliensis with PCR and sequencing methods. The recombinant protein with relative molecular mass about 38 000 was successfully induced by overexpressing HbADF in fission yeast. The cell morphology and the proportion of binucleate or coenocytic cells were further analyzed in the yeast overexpressing HbADF. The results showed that the length of yeast cells from overexpressing HbADF was markedly increased. In addition, it was observed that about 67% of the cells overexpressing HbADF were binucleate or coenocytic after induction. Real-time PCR analysis indicated that HbADF was regulated by 3% KI treatment and its expression varied with tapping panel dryness (TPD) stages and latex flow times. These results suggested that HbADF might play important roles in regulating TPD occurrence and latex flow in Hevea brasiliensis.