Water lettuce (Pistia stratiotes L.) is a free-floating perennial herbaceous plant with rosette leaves and a stem. Although this plant multiplies and has adverse effects on aquatic ecosystems, it can be used for biological purification of polluted water and production of valuable substances as a traditional medicine. In this study, we report a protocol to establish an in vitro micropropagation method based on direct shoot organogenesis from stem explants. In media comprising two types of basal medium and different growth regulators, multiple shoot organogenesis was observed on stems. The micropropagation method was most effective on Schenk and Hildebrandt (SH) medium with 1 mg/L indole-3-acetic acid and 2 mg/L 6-benzylaminopurine supplemented with 30 g/L sucrose, on which all explants produced multiple shoots. The shoots rooted spontaneously on solid half-strength Murashige and Skoog (MS) and SH media without growth regulators (1/2MSO and 1/2SHO). However, roots developed more vigorously in liquid media. Regenerated plants colonized and grew more rapidly in SH basal medium than in MS basal medium and produced 6–8 stolons within 2 weeks on 1/2SHO. In summary, we established a method for micropropagation in vitro through direct organogenesis of water lettuce, which shows the potential of water lettuce as a model aquatic plant for phytochemical and pharmacological research.