Mesenchymal stem cells (MSCs) from the Wharton’s jelly (WJ), a mucoid connective tissue of the umbilical cord having several therapeutic advantages are implicated in numerous clinical trials with target indications ranging from hematological to neurodevelopmental disorders. The effective cellular dose of WJ-MSCs derived from tissues is at a very low quantity, for application in pre-clinical/clinical trials; thus further commercialization relies on biomanufacturing to retain its proliferative multipotent state and therapeutic integrity. We have optimized the large-scale production of GMP-compliant xeno-free/serum-free human WJ-MSCs using a microcarrier-based stirred-tank bioreactor (5 L). A total yield of 2 × 109 (2.3 ± 400.34 ×109) cells/batch at a concentration of 5.0 × 105 cells/mL was achieved using a microcarrier platform in 14.0 ± 1.73 days corresponding to a 20 ± 1 fold expansion and harvest efficiency of 98.3 ± 4.5%. The upscaled WJ-MSCs were characterized and found to be of standard clinical grade. WJ-MSC inhibited phytohemagglutinin (PHA) mediated T-cell proliferation in vitro, the inhibitory potential could be enhanced by cytokine preconditioning of cells. A decrease in both the CD4 + and CD8 + populations and concomitant reduction in the CD25 + activation marker was noted. Display omitted •Large-scale production of human Wharton’s jelly (WJ) derived mesenchymal stem cells (MSCs) was achieved.•The microcarrier based stirred-tank bioreactor produced xeno-free/serum-free clinical grade cells.•A total yield of 2 × 109 (2.3 ± 400.34 ×109)/batch at a concentration of 5.0 × 105 cells/mL was achieved.•The cells had consistent batch to batch characteristics and the process is GMP-compliant.•The scaled-up cells have high demand and potential application in inflammatory/immunomodulatory clinical conditions.