Detarium microcarpum is a valuable tree species for fuelwood, timber, food and medicine in sub-Saharan Africa. However, its population is dwindling due to overexploitation, its seedlings’ low survival rate and slow growth. Vegetative propagation might enhance both survival and growth, but to date a successful clonal method does not exist for D. microcarpum. We conducted four experiments to examine the effects of propagation environment (high versus low humidity), cutting length and diameter, alignment of root segments (horizontal versus vertical) and distance from the root collar of donors on the regeneration ability of root segments collected from field-grown D. microcarpum trees in Burkina Faso. The size of root segments significantly affected their regeneration ability, while alignment had no effect. Sprouting was possible from 10 and 20-cm long segments of 15–60 mm diameter with 7–43% sprouting efficiency and multiple shoots while 5 cm long segments were unsuitable with 0–3% sprouting efficiency. Cuttings maintained at low humidity produced larger diameter sprouts than those in greenhouse. All cuttings showed strong polarity with most of the shoots developing at the proximal end. Rootlings from 20 cm root segments produced more new roots (0.62 ± 0.08 g) than those from 10 cm segments (0.34 ± 0.09 g), but they were similar for sprout and leaf growth. We conclude that lateral roots of field-grown mature trees can be used to produce rootlings in a nursery. Since this study is the first attempt to propagate D. microcarpum from root cuttings, further investigations are required to optimize the technique.