Melastoma malabathricum (L.) is a shrub that has potential uses in the fields of medicine and phytoremediation although further research on this species is warranted, including on its propagation, particularly in vitro culture. Here, research regarding the in vitro culture of M. malabathricum from internodes was conducted to determine its response on Murashige and Skoog (MS) modified medium supplemented with thidiazuron (TDZ) (1, 2, and 3 mg/L), 2,4-dichlorophenoxyacetic acid (2,4-D; 0.5 and 1 mg/L), or a combination of both. Our results showed that internodes responded by forming calluses on all treatment media except the medium supplemented with 0.5 mg/l 2,4-D and 3 mg/L TDZ. Compact calluses were formed upon supplementation with a single dose of TDZ (1, 2, and 3 mg/L) and a combination of growth regulators. Meanwhile, semi-compact calluses were formed upon supplementation with a single dose of 2,4-D (0.5 and 1 mg/L) and a combination of growth regulators. Generally, callus formation was observed on 5–19 days of culture. Internode explants cultured on medium supplemented with a single dose of 2,4-D showed optimal response, whereas those cultured on medium supplemented with the combination of TDZ and 2,4-D showed weak response.