Doxorubicin is an efficient chemotherapeutic reagent in the treatment of a variety of cancers. However, its underlying molecular mechanism is not fully understood and several severe side effects limit its application. In this study, the dynamic transcriptomic response of Saccharomyces cerevisiae cells to a doxorubicin pulse in a chemostat system was investigated to reveal the underlying molecular mechanism of this drug. The clustering of differentially and significantly expressed genes (DEGs) indicated that the response of yeast cells to doxorubicin is time dependent and may be classified as short-term, mid-term and long-term responses. The cells have started to reorganize their response after the first minute following the injection of the pulse. A modified version of Weighted Gene Co-expression Network Analysis (WGCNA) was used to cluster the positively correlated co-expression profiles, and functional enrichment analysis of these clusters was carried out. DNA replication and DNA repair processes were significantly affected and induced 60 minutes after exposure to doxorubicin. The response to oxidative stress was not identified as a significant term. A transcriptional re-organization of the metabolic pathways seems to be an early event and persists afterwards. The present study reveals for the first time that the RNA surveillance pathway, which is a post-transcriptional regulatory pathway, may be implicated in the short-term reaction of yeast cells to doxorubicin. Integration with regulome revealed the dynamic re-organization of the transcriptomic landscape. Fhl1p, Mbp1p, and Mcm1p were identified as primary regulatory factors responsible for tuning the differentially expressed genes.