The present study describes an effective, reproducible and rapid regeneration protocol for in vitro shoot development of the medicinally important, semi-aquatic plant Veronica anagallis-aquatica L. through mature nodal segments on Murashige and Skoog (MS) medium. The most effective medium for shoot regeneration was MS medium fortified with 0.5 mM 6-benzyladenine (BA), on which the maximum number (43.791.85) of shoots per explant having a shoot length of 5.090.25 cm were produced. The addition of auxins along with optimal concentration (0.5 mM) of BA did not further improve the regeneration capacity of the explants; instead, it resulted in an adverse effect. Nodal segments cultured in MS liquid medium without agar provided a better and early response for efficient shoot regeneration, and simultaneous rooting as compared to solidified medium. The optimal liquid medium comprised of MS'BA (0.5 mM) on which a maximum 20.791.76 shoots per explant were produced and grew healthier. Both half strength MS and MS basal medium supplemented with anaphthaleneacetic acid (NAA) responded equally for root induction. Rooted shoots were successfully transferred to field conditions with 80% survival. Histological studies revealed the de novo origin of shoots which would make this protocol suitable for transformation techniques.