Autophagy favors cell survival under hypoxia, and increasing evidence revealed that microRNAs regulate autophagy. We report here hypoxia increased the expression of miR-96 in prostate cancer cells, and miR-96 stimulated autophagy by suppressing MTOR. We found that inhibition of miR-96 abolished hypoxia-induced autophagy. Paradoxically, ectopic over-expression of miR-96 to a certain threshold, also abolished the hypoxia-induced autophagy. Further studies have shown that high levels of miR-96 inhibited autophagy through suppressing ATG7, a key autophagy-associated gene. Importantly, the miR-96 expression level threshold was determined, and the effects of miR-96 on autophagy on either side of the threshold were opposite. These data demonstrate hypoxia-induced autophagy is at least partially regulated by miR-96; miR-96 can promote or inhibit autophagy by principally inhibiting MTOR or ATG7 depending on the expression levels of miR-96. Our observation might reveal a novel regulatory mode of autophagy by microRNAs under hypoxia.