Purpose <p>In the present in vitro study, decellularized Wharton’s jelly (dWJ) scaffolds were loaded with kartogenin (KGN), and their chondrogenic effect on Wharton’s jelly-derived mesenchymal stem cells (WJMSCs) was evaluated.</p> Methods <p>WJ was extracted from fresh umbilical cords (UC), decellularized using SDS and Triton X-100, and characterized. dWJ scaffolds were loaded with KGN (25 and 50 µM) and characterized. WJMSCs were seeded on scaffolds and differentiated into the chondrogenic lineage. <i>Aggrecan</i>, <i>COLII</i>, <i>Sirt1</i>, and <i>SOX9</i> gene expression was assessed on days 7 and 21 using real-time PCR (<i>n</i> = 3).</p> Results <p>WJ was successfully decellularized, where cellular components were removed, and the structural integrity was preserved. dWJ scaffolds were highly porous, with porosities above 75% and pore sizes exceeding 100&#xa0;μm, which was favorable for KGN loading. Scaffolds possessed water uptake above 500% and gradually degraded over time. KGN loading slightly decreased scaffold porosity, water uptake, and degradation rates. 58% and 81% of KGN were loaded into the dWJ matrix in the KGN 25 and KGN 50 scaffolds, respectively, and released via a Fickian diffusion mechanism. dWJ scaffolds notably enhanced the expression of <i>aggrecan</i>, <i>COLII</i>, <i>Sirt1</i>, and <i>SOX9</i> genes in cultivated WJMSCs, with maximum 1.33 ± 0.1, 42.6 ± 6.4, 91.5 ± 6.3, and 59.9 ± 2.5 folds in dWJ.KGN 25 and 7.9 ± 0.4, 63 ± 5.5, 173.15 ± 10.9, and 117.3 ± 5.6 folds in dWJ.KGN 50 scaffolds on day 21.</p> Conclusion <p>dWJ and KGN-loaded dWJ scaffolds enhanced the chondrogenic differentiation of WJMSCs in vitro and could be regarded as a promising strategy for cartilage repair in the future. However, the current in vitro evidence is limited and requires further in vivo validation.</p> Graphical Abstract <p></p>