Drosophila RhoGAP18B was identified as a negative regulator of small GTPase in the behavioral response to ethanol. However, the effect of RhoGAP18B on cell migration is unknown. Here, we report that RhoGAP18B regulates the migration of border cells in Drosophila ovary. The RhoGAP18B gene produces four transcripts and encodes three translation isoforms. We use different RNAi lines to knockdown each RhoGAP18B isoform, and find that knockdown of RhoGAP18B-PA, but not PC or PD isoform, blocks border cell migration. Knockdown of RhoGAP18B-PA disrupts the asymmetric distribution of F-actin in border cell cluster and increases F-actin level. Furthermore, RhoGAP18B-PA may act on Rac to regulate F-actin organization. Our data indicate that RhoGAP18B shows isoform-specific regulation of border cell migration.