Background and Purpose: Andrographolide (AG) is a labdane diterpenoid herb, which is isolated from the leaves of Andrographis paniculate, and widely used for its potential medical properties. However, there are no reports on the effects of AG on the human suprapatellar fat pad of osteoarthritis patients. In the present study, our goal was to evaluate the innovative effects of AG on viability and Tri-lineage differentiation of human mesenchymal stem cells from suprapatellar fat pad tissues. Experimental approach: The effects of Andrographolide on viability and differentiation of human primary mesenchymal stem cells obtained from human suprapatellar fat pad tissues were evaluated by staining assay. Moreover, the effects on molecular expression were quantitatively measured by mRNA expression in real time PCR. Key Results: The results revealed that AG had no cytotoxic effects when the concentration was less than 12.5 µg/mL. Interestingly, AG had significantly enhanced, dose dependent, osteogenesis and chondrogenesis as evidenced by a significantly intensified stain for Alizarin Red S, Toluidine Blue and Alcian Blue. Moreover, AG can upregulate the expression of genes related to osteogenic and chondrogenic differentiation, including Runx2, OPN, Sox9, and Aggrecan in mesenchymal stem cells from human suprapatellar fat pad tissues. In contrast, AG suppressed adipogenic differentiation as evidenced by significantly diminished Oil Red O staining and expression levels for adipogenic-specific genes for PPAR-γ2 and LPL. Conclusions and Implications: These findings confirm that AG can specifically enhance osteogenesis and chondrogenesis of mesenchymal stem cells from human suprapatellar fat pad tissues. It has potential as a therapeutic agent derived from natural sources for regenerative medicine.