Although cellular and molecular mechanisms during the course of bone healing have been thoroughly investigated, the regulation of gene expression by microRNA during bone regeneration is still poorly understood. We hypothesized that nonunion formation is associated with different microRNA expression patterns and that target proteins of these microRNAs are differently expressed in callus tissue of nonunions compared to physiologically healing bones. In a well-established femoral osteotomy model in CD-1 mice osteotomies were induced which result either in healing or in nonunion formation. MicroRNA and target protein expression was evaluated by microarray, quantitative real-time polymerase chain reaction (qrt-PCR) and Western blot. Microarray analyses demonstrated 44 microRNAs to be relevant for nonunion formation compared to physiological bone healing. In nonunions qrt-PCR could validate a higher expression of microRNA-140-3p and microRNA-140-5p. This was associated with a reduced expression of Dnpep and stromal cell-derived factor (SDF)-1α, which are both known to be target proteins of microRNA-140 and also to be involved in the process of bone healing. These data suggest that an increased expression of microRNA-140-3p and microRNA-140-5p markedly contributes to the development of nonunions, most probably by affecting bone morphogenetic protein (BMP)-2 function during the early stage of healing due to a reduced SDF-1α expression.