Prorocentrum donghaiense is one of the main red tide organisms in China’s coastal waters. It is characterized by frequent outbreaks, a large area of influence, and serious harm. The cell cycle is an important biological process that regulates cell division. It can be affected by environmental factors, resulting in changes in the growth rate of phytoplankton. Therefore, it is valuable to investigate the differences in the cell cycle progression of P. donghaiense with different proliferation rates to help understanding its proliferation characteristics. The cell cycle pattern of P. donghaiense has been preliminary explored. Genes and proteins related to the cell cycle have been isolated and identified using omics approaches. However, there is a lack of research on the characteristics of the cell cycle with different proliferation rates, as well as the molecular mechanism. Our previous studies demonstrated that the growth rate of P. donghaiense varies under different environmental conditions. To compare the cell cycle progression of P. donghaiense with different proliferation rates and understand its molecular mechanism, we selected two experimental groups with slower (group 3) and faster (group 16) proliferation rates, and analyzed them using flow cytometry and RNA sequencing technology. The results showed that group 3 cells had a shorter S phase and were partially blocked in G2/M phase. A total of 149219 unigenes were obtained, of which 6081 were annotated in the NR, GO, KEGG, eggNOG, Swiss-Prot, and Pfam databases. A total of 114358 unigenes were classified into three terms, namely, cellular components, molecular functions, and bioprocesses, with 57 categories in the GO database. The 30554 unigenes annotated in the eggNOG database were classified into 25 categories. GO classification and pathway enrichment results of differentially expressed genes (DEGs) between the two experimental groups showed that the most important DEGs were related to microtubules and the cytoskeleton. We hypothesized that the differences in cell cycle progression between the two experimental groups were caused by differences in the dynamic changes of microtubules. Among the annotated unigenes, 49 encoding cyclins, 74 encoding cyclin-dependent kinases (CDKs), and 26 encoding cell division cycle (CDC) proteins were related to cell cycle control. Ten of these comprising one CYCA, one CYCB, one CYCU, one CDK1, four CDK2, and one Cdc48 genes were DEGs. The cyclinA-CDK2 and CDK1-cyclinB complexes participate in the cellular G2/M phase transition. Cells need to retain sufficient levels of CDK activity to maintain the transcription of G2-specific genes and retain their competence to recover from G2 arrest. Since cells in group 3 were partially arrested in G2/M, the transcriptional levels of CDKs, cylinA, and cyclinB were higher in group 3 than in group 16. As Cdc48 is involved in cellular DNA replication, the transcription level of Cdc48 was significantly higher in group 16 than in group 3, which explained why the S phase duration was longer in group 16 than in group 3. In summary, the S phase duration and whether the G2/M phase transition is blocked may decide the rate of P. donghaiense cell proliferation, and one CYCA, one CYCB, one CYCU, two CDK1, four CDK2, and one Cdc48 genes identified from the transcriptome analysis played important roles in cell cycle regulation.