Background　Adipose　tissue-derived　mesenchymal　stem　cells　(ADSCs)　are　promising　candidates　for　regenerative　medicine.　However,　long-term　in　vitro　passaging　leads　to　stemness　loss　and　cell　senescence　of　ADSCs,　resulting　in　failure　of　ADSC-based　therapy.　Methods　In　this　study,　ADSCs　were　treated　with　low　dose　of　antioxidants　(reduced　glutathione　and　melatonin)　with　anti-aging　and　stem　cell　protection　properties　in　the　in　vitro　passaging,　and　the　cell　functions　including　stem　cell　senescence,　cell　migration,　cell　multidirectional　differentiation　potential,　and　ROS　content　were　carefully　analyzed.　Results　We　found　that　GSH　and　melatonin　could　maintain　ADSC　cell　functions　through　reducing　cell　senescence　and　promoting　cell　migration,　as　well　as　by　preserving　stemness　and　multidirectional　differentiation　potential,　through　inhibiting　ROS　generation　during　long-term　expansion　of　ADSCs.　Conclusions　Our　results　suggested　that　antioxidant　treatment　could　efficiently　prevent　the　dysfunction　and　preserve　cell　functions　of　ADSCs　after　long-term　passaging,　providing　a　practical　strategy　to　facilitate　ADSC-based　therapy.