SUMO (Small ubiquitin-related modifier) modification (SUMOylation) is a highly dynamic post-translational modification (PTM) playing important roles in tissue development and disease progression. However, its function in adult stem cell maintenance is largely unknown. Here we report the function of SUMOylation in somatic cyst stem cells (CySCs) self-renewal in adult Drosophila testis. The SUMO pathway cell-autonomously regulates CySCs maintenance. Reduction of SUMOylation promotes premature differentiation of CySCs and impedes the proliferation of CySCs, which finally reduce the number of CySCs. Consistently, CySC clones carrying mutation of the SUMO conjugating enzyme are rapidly lost. Furthermore, inhibition of SUMO pathway phenocopies the disruption of Hedgehog (Hh) pathway, and can block the promoted proliferation of CySCs by Hh activation. Importantly, SUMO pathway directly regulates the SUMOylation of Hh pathway transcriptional factor, Cubitus interruptus (Ci), which is required for promoting CySCs proliferation. Thus, we conclude that SUMO directly targets Hh pathway and regulates CySCs maintenance in adult Drosophila testis.