Abstract: Plantlets regeneration were achieved in callus culture of leaf segments from five days old field grown seedlings of Citrulus lanatus Thumb. Callus induction and plant regeneration at various frequencies were observed using different concentrations and combinations of growth regulators. The highest percentage of callus induction was observed in MS medium supplemented with 2.5 mg L-1 2, 4-D. After transfer of this callus on MS medium supplemented with 1.0 mg L-1 BA+0.2 mg L-1 NAA induce adventitious shoots and developed into plant by further subculture in the same medium. NAA (0.1 mg L-1) was found effective in the production of root. Plantlets were acclimatized and subsequently transferred to the field. Survival of the plantlets under ex vitro condition was 80%.