Down-regulation of microRNA-21 (miR-21) can induce cell apoptosis and reverse drug resistance in cancer treatments. In this study, we explored the most effective schedule of the miR-21 inhibitor (miR-21i) and Temozolomide (TMZ) combined treatment in human glioma cells. Three tumor cell lines, U251 phosphatase and tensin homologue (PTEN) mutant, LN229 (PTEN wild-type), and U87 (PTEN loss of function), were subjected to evaluate the antitumor effects of deigned treatments (a predose of miR-21i for 4/8 h and then a subsequent TMZ treatment, a predose of TMZ for 4/8 h and then a subsequent miR-21i treatment, or a concomitant treatment) in vitro. A synergistic antiproliferative and proapoptotic activity was only obtained in U251 and U87 cells when a predose was administered for 4 h before the treatment of the other therapeutic agent, while the best antitumor effect in LN229 cells was achieved by using the concomitant treatment. Our data indicate that the effect of sequence and timing of administration is dependent on the PTEN status of cell lines. The best suppression effect was achieved by a maximal inhibition of STAT3 and phosphorylated STAT3, in PTEN loss of function cells. Our results reveal that both the sequence and the timing of administration are crucial in glioma combination therapy.