Polyvinyl alcohol (PVA) hydrogels are well-known biomimetic 3D systems for mammalian cell cultures to mimic native tissues. Recently, several biomolecules were intended for use in PVA hydrogels to improve their biological properties. However, retinol, an important biomolecule, has not been combined with a PVA hydrogel for culturing bone marrow mesenchymal stem (BMMS) cells. Thus, for the first time, the effect of retinol on the physicochemical, antimicrobial, and cell proliferative properties of a PVA hydrogel was investigated. The ability of protein (3.15 nm) and mineral adsorption (4.8 mg/mL) of a PVA hydrogel was improved by 0.5 wt.% retinol. The antimicrobial effect of hydrogel was more significant in S. aureus (39.3 mm) than in E. coli (14.6 mm), and the effect was improved by increasing the retinol concentration. The BMMS cell proliferation was more upregulated in retinol-loaded PVA hydrogel than in the control at 7 days. We demonstrate that the respective in vitro degradation rate of retinol-loaded PVA hydrogels (RPH) (75–78% degradation) may promote both antibacterial and cellular proliferation. Interestingly, the incorporation of retinol did not affect the cell-loading capacity of PVA hydrogel. Accordingly, the fabricated PVA retinol hydrogel proved its compatibility in a stem cell culture and could be a potential biomaterial for tissue regeneration.