Human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes (Exo) have been frequently investigated for disease control. This study was designed to explore the effects of hucMSC-Exo carrying lncRNA family with sequence similarity 99-member B (Exo-lncRNA FAM99B) on hepatocellular carcinoma (HCC) cell behaviour. The expression of lncRNA FAM99B in HCC cells was measured by reverse-transcription quantitative polymerase chain reaction. Protein levels of exosomal markers were quantified using western blotting. Flow cytometry analyses were performed to detect surface markers of hucMSCs and to measure the effects of Exo-lncRNA FAM99B on HCC cell cycle progression and cell apoptosis. Nanoparticle tracking analysis was used to measure the particle size of the exosomes. Additionally, cell viability was evaluated using methyl thiazolyl tetrazolium assays, and Transwell assays were performed to measure cell migration and invasion. Xenograft tumor models were established to explore the role of Exo-lncRNA FAM99B in vivo. Experimental results revealed that lncRNA FAM99B was downregulated in HCC cell lines, and low level of FAM99B is associated with poor survival rates in patients with HCC according to bioinformatics analysis. HucMSCs were identified in a good morphology with positively expressed CD105, CD29, and CD44 as well as negatively expressed CD31, CD14, and HLA-DR. High protein levels of exosomal markers (Alix, CD63 and TSG101) identified the existence of HucMSC-Exo. Importantly, the hucMSCs-Exo could enter HCC cells and exerted a suppressive effect on malignant cell activities. Moreover, overexpression of Exo-lncRNA FAM99B enhanced cell cycle arrest and cell apoptosis while suppressing cell viability, migration, and invasion in HCC. Exo-siRNA-FAM99B exerted the opposite effects on HCC cell process. In vivo experiments verified that Exo-lncRNA FAM99B inhibited tumorigenesis in HCC. In summary, lncRNA FAM99B derived from hucMSC-Exo inhibited malignant cellular phenotypes and tumorigenesis in HCC, which might provide a novel therapeutic strategy for HCC treatment.© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.