Mesenchymal stem cells (MSCs) are an important source of cells for bone regeneration. Although the utilization of MSCs along with growth factors and scaffolds is a beneficial clinical approach for bone tissue engineering, there is need for improvement on the effectiveness of MSC osteogenesis and differentiation. Rutin is a natural flavonoid and a major component for cell proliferation and bone development. However, studies on the mechanism through which rutin regulates osteogenesis and MSC differentiation are limited. Therefore, this study aimed to investigate the effect and mechanisms of rutin on osteogenic differentiation of MSCs. MSCs were extracted from umbilical cords and treated with rutin, followed by the examination of osteogenesis-related markers. Rutin treatment promoted the differentiation of MSCs towards the osteogenic lineage rather than the adipogenic lineage and increased the expression of osteogenic markers. RNA sequencing and bioinformatic analysis indicated that rutin regulated p53, a key gene in regulating the osteogenic differentiation of MSCs. Additionally, cellular experiments showed that rutin-induced decrease in p53 expression increased the formation of extracellular matrix (ECM) by promoting p65 phosphorylation and caspase-3 cleavage. Conclusively, this study demonstrates the importance of rutin in osteogenesis and indicates that rutin possesses potential pharmaceutical application for bone regeneration and bone tissue engineering.