Malanga (Colocasia esculenta) is a plant genetic resource that requires biotechnological strategies for conservation and propagation. One time-, labor-, and space-saving option is in vitro conservation and regeneration. The objective of this study was to develop a protocol for in vitro regeneration and conservation of germplasm of C. esculenta var. criolla. For conservation through minimal growth, we assessed several concentrations of Murashige and Skoog (MS) medium (one-third, one-half, and three-quarter strength), the growth retardant ancymidol (0, 1, 2, and 3 mg·L−1), and the osmoregulator polyethylene glycol (PEG-8000 mw) at different concentrations (0, 10, 20, and 30 g·L−1). For in vitro conservation, the percent survival, shoot number and length, and number of leaves and roots per explant were evaluated after 24 weeks. For in vitro regeneration, different concentrations of thidiazuron (TDZ: 0, 0.5, 1, 1.5, and 2 mg·L−1) and 6-benzylaminopurine (BAP; 0, 1, 2, 3, and 4 mg·L−1) were evaluated. After 4 weeks of cultivation, the percent response, shoot number, and number of leaves per explant were recorded. During in vitro conservation, it was noted that the treatment including 2 mg·L−1 ancymidol resulted in a retarded development, without affecting the survival of the C. esculenta germplasm. With regard to shoot regeneration, 7.60 shoots per explant were obtained using 2 mg·L−1 TDZ. Finally, 98% survival was achieved during the acclimatization process. This study will contribute to the establishment of genetic improvement programs through in vitro conservation and propagation of this valuable plant genetic resource.