<p>Genetically transformed root (TR) clones (rhizoclones) were developed of an ethnomedicinally important plant species, <i>Sida acuta</i> mediated by <i>Rhizobium rhizogenes</i>. Various key factors (bacterium × plant × culture environment) were optimized for the transformation event per se and hairy root growth. RT-PCR amplification demonstrated transcription activity of opine biosynthetic genes and production of opines in rhizoclones was confirmed. Anti-hyperglycemic activity of the methanolic/aqueous extracts of a selected <i>rol</i>B ORF-positive fast-proliferating TR clone was investigated <i>vis-á-vis</i> natural roots (NR) employing streptozotocin-induced diabetic Wistar rats. Compared to root extracts of naturally occurring donor plants, aqueous extract of the TR clone caused a more pronounced reducing effect on fasting blood glucose level and normalization of biochemical indices (serum and urine) as hepatic/renal functional markers. Metabolic attenuation was evident in respect of insulin and hepatic glycogen content as well as biochemical levels of blood serum, liver-specific enzymes and urine-specific biochemicals. Aqueous TR extracts, and the purified β-sitosterol isolate from such extracts, at optimal levels, demonstrated anti-hyperglycemic activity to a considerable extent, which was comparable with that of the commercial drug glibenclamide towards restitution of biochemical homeostasis in the diabetic rats. Our finding on fast-renewable transformed root culture extract, capable of a noteworthy glycemic control in diabetic rats, ushers a possibility of designing and developing potent bioactive anti-diabetic drug(s) to be implicated in human healthcare.</p> Graphical abstract <p></p>