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Stem cells have the potential to maintain undifferentiated state and differentiate into specialized cell types. Despite numerous progress has been achieved in understanding stem cell self-renewal and differentiation, many fundamental questions remain unanswered. In this study, we identify dRTEL1, the Drosophila homolog of Regulator of Telomere Elongation Helicase 1, as a novel regulator of male germline stem cells (GSCs). Our genome-wide transcriptome analysis and ChIP-Seq results suggest that dRTEL1 affects a set of candidate genes required for GSC maintenance, likely independent of its role in DNA repair. Furthermore, dRTEL1 prevents DNA damage-induced checkpoint activation in GSCs. Finally, dRTEL1 functions to sustain Stat92E protein levels, the key player in GSC maintenance. Together, our findings reveal an intrinsic role of the DNA helicase dRTEL1 in maintaining male GSC and provide insight into the function of dRTEL1. Author summary: Adult tissue homeostasis is maintained by its resident tissue-specific stem cells. The maintenance of stem cells depends on niche activity (the so-called tissue-specific microenvironment) as well as stem cell intrinsic factors. In a genetic screen, we identify dTREL1 as an intrinsic factor required for the maintenance of Drosophila male germline stem cells (GSCs). Loss of dRTEL1 activity leads to premature loss of male GSCs. Our study shows that dRTEL1 likely functions through multiple downstream genes to promote GSC maintenance. Further results indicate that dRTEL1 also prevents activation of the DNA damage response pathway in GSCs for its maintenance. These factors likely converge on the regulation of Stat92E, a well-studied intrinsic factor for GSC maintenance. [ABSTRACT FROM AUTHOR]