Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and one of the prominent causes of cancer mortality, leading to approximately 780,000 deaths per year worldwide. Down-regulation of microRNA-125b (miR-125b) is a prognostic indicator in HCC patients. Conversely, over-expression of miR-125b in HCC cells induces cell cycle arrest, inhibits proliferation, migration and invasion. Extracellular vesicles (EVs) function as intercellular messengers transferring proteins, RNAs, DNAs, carbohydrates, and lipids. Since EVs protect their cargo from degradation, delivery of therapeutic bioactive molecules, in particular miRNAs, through EVs represents an innovative avenue for cancer therapy. In this study, we evaluated a replacement strategy for the treatment of HCC via delivery of EVs secreted from human adipose tissue-derived mesenchymal stromal/medicinal signaling cells (ASCs) genetically modified with a lentiviral vector expressing miR-125b with a specific ExoMotif sequence tag to enhance the loading into extracellular vesicles. In particular, we determined that the delivery of miR-125b-loaded EVs produced in engineered ASCs specifically reduces HCC cell proliferation in vitro modulating a series of miR-125b targets, which belong to the p53 signaling pathway. This proof-of-concept study supports the development of innovative therapeutic strategies for HCC via EV-mediated miRNA delivery.