Transcriptome analysis has played an essential role for revealing gene expression and the complexity of regulations at transcriptional level. RNA-seq is a powerful tool for transcriptome profiling, which uses deep-sequencing technologies to directly determine the cDNA sequence. Here, we utilized RNA-seq to explore the transcriptome of Mycobacteriummarinum (M. marinum), which is a useful model to study the pathogenesis of Mycobacterium tuberculosis (Mtb). Two profiles of exponential and early stationary phase cultures were generated after a physical ribosome RNA removal step. We systematically described the transcriptome and analyzed the functions for the differentiated expressed genes between the two phases. Furthermore, we predicted 360 operons throughout the whole genome, and 13 out of 17 randomly selected operons were validated by qRT-PCR. In general, our study has primarily uncovered M. marinum transcriptome, which could help to gain a better understanding of the regulation system in Mtb that underlines disease pathogenesis.