Accelerated root formation is necessary for successful cuttage in a number of woody plants, such as mulberry (Morus alba L.). However, little information regarding the molecular mechanisms involved in this type of root formation is available. Here, we compared transcriptome changes during different root formation periods (0, 4, and 8 days) by conducting high-throughput sequencing to understand the molecular mechanisms of root formation in mulberry softwood cuttings. Approximately 88.2 million, 88.2 million, and 52.8 million transcriptome sequencing reads were obtained for the three time periods, 0, 4, and 8 days, respectively. These reads were then assembled into 61,687 (0 day), 61,651 (4 days), and 59,457 (8 days) contigs by utilizing the de novo method, and a large number of differentially expressed genes were identified at various developmental stages. These assembled contigs were annotated using Kyoto Encyclopedia of Genes and Genomes (KEGG), as well as the simple sequence repeat (SSR). Mulberry had a complex response to adventitious rooting. Genes that might be associated with mulberry stem cutting root formation were also identified here. Furthermore, the reliability of the sequencing results was validated by performing quantitative real-time polymerase chain reaction (qRT-PCR). These results provide a comprehensive background of the molecular biology of mulberry development, in particular regarding its stem cutting root formation development.