Medicago truncatula is used widely as a model system for studies of root symbioses, interactions with parasitic nematodes and fungal pathogens, as well as studies of development and secondary metabolism. In Medicago truncatula as well as other legumes, RNA interference (RNAi) coupled with Agrobacterium rhizogenes-mediated root transformation, has been used very successfully for analyses of gene function in roots. One of the major advantages of this approach is the ease and relative speed with which transgenic roots can be generated. There are several methods, both for the generation of the RNAi constructs and the root transformation. Here we provide details of an RNAi and root transformation protocol that has been used successfully in M. truncatula and which can be scaled up to enable the analysis of several hundred constructs.