Neuronal apoptosis is one of the prominent features involved in spinal cord injury (SCI). MicroRNAs (miRNAs) are small non-coding RNAs that functions in a variety of cellular processes including apoptosis. MiRNAs have been implicated as effectors of SCI. However, role of miRNAs in SCI-associated neuronal apoptosis remains to be investigated. A number of bioinformatics approaches have suggested Mcl-1 and BH3-only family genes as potential downstream targets regulated by miR-20a and miR-29b, respectively. To determine whether miR-20a and miR-29b play a role in neuronal apoptosis of SCI by regulating those genes, we transfected Neuro-2A neuroblastoma cells with mimic and inhibitor for the two miRNAs. The miR-20a mimic decreased Mcl-1 expression and the miR-29b mimic reduced the expression of Bad, Bim, Noxa and Puma. The repressor role of miR-20a and miR-29b is confirmed by the transfection of Neuro-2A cells with their inhibitor. Moreover, miR-20a mimic or miR-29b inhibitor attenuated Neuro-2A cell viability and co-transfection of both further diminished the viability of these cells. The in vitro effects of miR-20a and miR-29b on neuronal apoptosis were corroborated by the in vivo studies. Injection of miR-20a mimic or miR-29b inhibitor into the lesion of the injured spinal cord rescued the neuronal death and co-injection of both completely abolished SCI-induced apoptosis. In conclusion, altered expression of miR-20a and miR-29b may cooperatively contribute to the neuronal cell death of SCI through down-regulating anti-apoptotic myeloid cell leukemia sequence-1 (Mcl-1) and up-regulating pro-apoptotic BH3-only proteins.