Human multipotent mesenchymal stromal cells (hMSCs) are promising candidates for clinical application due to their intrinsic immunomodulatory properties and their ability to migrate towards inflammatory and tumour sites. To improve their clinical potential, hMSCs can be genetically engineered to express, for example, therapeutic transgenes or deliver oncolytic viruses to target sites. For such approaches, vectors based on human adenovirus type 5 (HAdV-5) are valuable tools. But, transduction of hMSCs with unmodified HAdV-5 vectors is very inefficient. We identified several positively charged transduction enhancers that, using a very simple protocol, impressively increased HAdV-5-mediated gene transfer into bone marrow- and adipose tissue-derived hMSCs. Based on this finding, we introduced positively charged amino acids into the viral capsid by genetic engineering. While most modifications of adenoviral capsids focus on the protruding Fiber, we altered distinct surface-exposed amino acids in Hexon, the most abundant protein in the adenoviral capsid. Strikingly, one of the generated vectors (HAdV-5-HexPos3) transduced hMSCs more efficiently than any other adenovirus-based strategy published so far. We assume that transduction enhancers and HAdV-5-HexPos3 are valuable tools for engineering hMSCs to enable a plethora of applications. For example, hMSCs engineered to express firefly luciferase could be accurately tracked upon intravenous administration to mice. Moreover, we show that high-level expression of therapeutic transgenes is feasible, and efficient virus replication of wild-type or conditionally replicating HAdV-5 vectors in hMSCs can be achieved, which demonstrates the potential for clinical application.