AbstractTGF-β/Activin induces epithelial-to-mesenchymal transition and stemness in pancreatic ductal adenocarcinoma (PDAC). However, the microRNAs (miRNAs) regulated during this response have remained yet undetermined. Here, we show that TGF-β transcriptionally induces MIR100HG lncRNA, containing miR-100, miR-125b and let-7a in its intron, via SMAD2/3. Interestingly, we find that although the pro-tumourigenic miR-100 and miR-125b accordingly increase, the amount of anti-tumourigenic let-7a is unchanged, as TGF-β also induces LIN28B inhibiting its maturation. Notably, we demonstrate that inactivation of miR-125b or miR-100 affects the TGF-β-mediated response indicating that these miRNAs are important TGF-β effectors. We integrate AGO2-RIP-seq with RNA-seq to identify the global regulation exerted by these miRNAs in PDAC cells. Transcripts targeted by miR-125b and miR-100 significantly overlap and mainly inhibit p53 and cell–cell junctions’ pathways. Together, we uncover that TGF-β induces an lncRNA, whose encoded miRNAs, miR-100, let-7a and miR-125b play opposing roles in controlling PDAC tumourigenesis.