Abstract: An investigation was undertaken at Plant Biotechnology Laboratory, Biotechnology Discipline, Khulna University, Khulna to study the performance of different carbon sources and growth regulators on in vitro plantlet formation of Thankuni (Centella asiatica L.) from nodal segment. Among the carbon sources, sucrose showed the best results on shoot formation in presence of BA or Kn. In these cases the sucrose along with BA obtained 100% and sucrose along with Kinetin obtained 90% shoot formation, respectively. Maximum shoot length was obtained (2.67±0.059 cm) in sucrose containing BA medium. Other carbon sources showed less performance than sucrose both on BA and Kn containing media. Interestingly maltose showed the highest number of shoot/explant (4.0±0.0017) in presence of BA. In combination of BA+Kn, the percentage of shoot formation was higher (90%) in presence of sucrose, but the number of shoot/explant was maximum (3±0.035) in presence of maltose. In root formation, sucrose was found superior than other carbon sources used in present experiment. In this case IBA obtained the highest percentage of root (80%) with maximum length (1.5±0.019 cm), whereas, gur and maltose did not produce any root.