Siegesbeckia orientalis L. was identified as a novel Cd-hyperaccumulator and valuable phytoremediation material. However, the molecular mechanisms underlying Cd accumulation in S. orientalis are largely unknown. In this study, RNA-Seq analysis was performed to study the Cd-accumulating mechanisms in its roots with or without Cd treatment. The RNA-seq analysis generated 312 million pairs of clean reads and 78G sequencing data. De novo transcriptome assembly produced 355,070 transcripts with an average length of 823.59 bp and 194,207 unigenes with an average length of 605.68 bp. Comparative transcriptome analyses identified a large number of differentially expressed genes in roots under Cd stress, and functional annotation suggested that S. orientalis utilizes various biological pathways involving many gene networks working simultaneously to cope with the stress. This study revealed that four biological pathways were mainly involved in S. orientalis tolerance to Cd stress, including reactive oxygen species scavenging, phenylpropanoid biosynthesis pathway, Cd absorption and transport, and ABA signaling pathway. The genes related to photosynthesis and heavy metal transport are likely the potential candidates and could be further investigated to determine their roles in Cd tolerance in S. orientalis roots. These findings will be useful to understand the Cd accumulation mechanisms in S. orientalis and facilitate the study of phytoremediation at the molecular level in plants.