Mitragyna speciosa is a widely used medicinal plant that is commonly used for its morphine-like effect sin folklore medicine in Thailand and Malaysia due to its ability to reduce pain and ameliorate withdrawal signs after cessation of opioid abuse. The aim of the present study was to determine and compare the relative safety and therapeutic indices of M. speciosa alkaloid extract and its major component, mitragynine. An alkaloid extract (20–400 mg/kg) from the leaves of M. speciosa, as well as mitragynine (4.2–84 mg/kg), was orally administered to mice; dose–response relationship, ED50 and LD50 values, as well as the therapeutic index (TI), for the two substances were determined and compared with that of morphine (2.5–10 mg/kg, s.c.). The results showed a significant dose-dependent response in both extract (50 mg/kg onward) and mitragynine (10.5 mg/kg) with a higher potency of mitragynine than that of the extract. Although the LD50 for the extract (591 mg/kg) was higher than that of mitragynine (477 mg/kg), the TI for mitragynine was wider than that of the extract (21:3). The present study indicated that mitragynine is relatively safer when compared with the alkaloid extract of M. speciosa in mice.