Paclitaxel (Taxol) is an effective chemotherapeutic agent for treatment of cancer patients. Despite impressive initial clinical responses, the majority of patients eventually develop some degree of resistance to Taxol-based therapy. The mechanisms underlying cancer cells resistance to Taxol are not fully understood. MicroRNA (miRNA) has emerged to play important roles in tumorigenesis and drug resistance. However, the interaction between the development of Taxol resistance and miRNA has not been previously explored. In this study we utilized a miRNA array to compare the differentially expressed miRNAs in Taxol-resistant and their Taxol-sensitive parental cells. We verified that miR-125b, miR-221, miR-222, and miR-923 were up-regulated in Taxol-resistant cancer cells by real-time PCR. We further investigated the role and mechanisms of miR-125b in Taxol resistance. We found that miR-125b was up-regulated in Taxol-resistant cells, causing a marked inhibition of Taxol-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to Taxol in cancer cells. Moreover, we demonstrated that the pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) is a direct target of miR-125b. Down-regulation of Bak1 suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol. Restoring Bak1 expression by either miR-125b inhibitor or re-expression of Bak1 in miR-125b-overexpressing cells recovered Taxol sensitivity, overcoming miR-125-mediated Taxol resistance. Taken together, our data strongly support a central role for miR-125b in conferring Taxol resistance through the suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming Taxol resistance in a number of different tumor histologies.