SUMO (Small ubiquitin-related modifier) modification (SUMOylation) is a highly dynamic post-translational modification playing important roles in tissue development and disease progression via regulating a wide range of cellular processes.However, its function in adult stem cell is rarely reported.The adult Drosophila testis turns out to be a powerful genetic system to study stem cell maintenance.Although several cellular pathways have been demonstrated to play important roles in sustaining the stem cell populations, there is a pressing need to seek for novel regulators considering the complex genetic networks.In this study,SUMO pathway was identified to regulate somatic cyst stem cells (CySCs) in Drosophila adult testis.Blockage of SUMOylation reduced the number of CySCs,while artificially sustained SUMOylation expanded the CySCs pool, respectively similar to the phenotypes induced by suppression or activation of Hedgehog (Hh) signaling pathway.Besides, SUMO pathway positively modulated the Hh signaling activity and impeding SUMOylation abrogated the CySCs over-proliferation caused by hyper-activation of Hh signaling.Mechanistically, we found that Cubitus interruptus (Ci, the transcription factor of Hh pathway) could be SUMOylated and SUMO conjugation-deficient mutants of Ci loss the ability to promote CySCs proliferation.In summary, we identified a novel role of the SUMO pathway in CySCs maintenance, partially through regulating the Hh signaling activity by directly targeting Ci for SUMOylation.