Myeloid cell leukemia 1 (MCL1) is a key anti-apoptotic protein belonging to the BCL-2 protein family. To preserve normal cellular homeostasis, cells must maintain strict control over MCL1 expression. Overexpression of MCL1 has been identified as a key contributor to tumorigenesis, and further enables resistance to a number of anti-cancer chemotherapies. Thus, there is an ongoing interest to develop selective MCL1 inhibitors. In order to better target MCL1, it is essential to understand the molecular mechanisms that regulate MCL1 expression in cells. While MCL1 expression is tightly controlled by multiple mechanisms, the post-transcriptional regulation of MCL1 mRNA is poorly studied. Our previous work identified that polypyrimidine tract binding protein 1 (PTBP1) binds to MCL1 mRNA and represses MCL1 expression by destabilizing MCL1 mRNA. In this report, we show that PTBP1 modulates MCL1 expression by regulating the microRNA (miRNA) direction of the miRNA-induced silencing complex (miRISC) to MCL1. We demonstrate that PTBP1 enhances miR-101-guided AGO2 interaction with MCL1, thereby regulating miR-101-induced apoptosis and clonogenic cell survival inhibition in cells. Taken together, not only do these studies expand our understanding on the regulation of MCL1, they also demonstrate that PTBP1 and miRNAs can function cooperatively on a shared target mRNA.