Myotubularin (MTM) and myotubularin-related (MTMR) lipid phosphatases catalyze the removal of a phosphate group from certain phosphatidylinositol derivatives. Because some of these substrates are required for macroautophagy/autophagy, during which unwanted cytoplasmic constituents are delivered into lysosomes for degradation, MTM and MTMRs function as important regulators of the autophagic process. Despite its physiological and medical significance, the specific role of individual MTMR paralogs in autophagy control remains largely unexplored. Here we examined two Drosophila MTMRs, EDTP and Mtmr6, the fly orthologs of mammalian MTMR14 and MTMR6 to MTMR8, respectively, and found that these enzymes affect the autophagic process in a complex, condition-dependent way. EDTP inhibited basal autophagy, but did not influence stress-induced autophagy. In contrast, Mtmr6 promoted the process under nutrient-rich settings, but effectively blocked its hyperactivation in response to stress. Thus, Mtmr6 is the first identified MTMR phosphatase with dual, antagonistic roles in the regulation of autophagy, and shows conditional antagonism/synergism with EDTP in modulating autophagic breakdown. These results provide a deeper insight into the adjustment of autophagy.Abbreviations: Atg, autophagy-related; BDSC, Bloomington Drosophila Stock Center; DGRC, Drosophila Genetic Resource Center; EDTP, Egg-derived tyrosine phosphatase; FYVE, zinc finger domain from Fab1 (yeast ortholog of PIKfyve), YOTB, Vac1 (vesicle transport protein) and EEA1 cysteine-rich proteins; LTR, LysoTracker Red; MTM, myotubularin; MTMR, myotubularin-related; PI, phosphatidylinositol; Pi3K59F, Phosphotidylinositol 3 kinase 59F; PtdIns3P, phosphatidylinositol-3-phosphate; PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate; PtdIns5P, phosphatidylinositol-5-phosphate; ref(2)P, refractory to sigma P; Syx17, Syntaxin 17; TEM, transmission electron microscopy; UAS, upstream activating sequence; Uvrag, UV-resistance associated gene; VDRC, Vienna Drosophila RNAi Center; Vps34, Vacuolar protein sorting 34.