Abstract: Bioassay guided-fractionation of Mitragyna ciliata Aubrev and Pellegr (Rubiaceae) ethanolic root extract at 100 mg kg-1 in T. brucei infected rats indicated that the bioactive constituent reside is present in the butanol fraction (inhibition = 66.61%). In vitro investigation of the extract revealed that it had low (25.55%) antioxidative property. Chemical analysis of the active fraction showed that it consists of alkaloids. The extract`s fraction had no effect on the heamatological profile of treated rats which remained consistent with the major characteristics of trypanosomiasis-anaemia, leucocytopaenia and thrombocytopaenia. Results of the in vivo evaluation of calcium concentration showed a significant difference (p<0.05), between the active butanol fraction (2.53 ± 0.036 mmol L-1) and the untreated/infected (17.79 ± 0.034 mmol L-1). A correlation existed between calcium concentration and parasiteamia in the active fraction (r = 0.40, p = 0.488) and the infected/untreated (r = -0.60, p = 0.29). These observations suggest that the active agent had an effect on the calcium metabolism in the animals which was deleterious to the parasites.