Cyanobacterial blooms are a worldwide phenomenon that arise due to nutrient-rich conditions. Specific species comprising cyanobacterial blooms produce toxins detrimental to human and animal health. Of particular concern is microcystin, a potent hepatotoxin produced primarily by the unicellular species Microcystis aeruginosa. Understanding the key cellular processes involved in microcystin production likely will inform on strategies for toxic species control, through the establishment of novel molecular targets to inhibit toxin synthesis and suppress toxic species proliferation. Despite several investigations from different groups, comprehensive information regarding the effect of microcystin on cellular processes is lacking. Recent advances in proteomic technologies allow the comprehensive coverage of the cell proteome, that is, the proteins expressed at a certain point in time. In this study, we employ a previously established fosmid containing the M. aeruginosa biosynthetic gene cluster expressed heterologously in Escherichia. coli, a construct employed in the rapid biosynthesis of microcystin. Absolute quantification of proteins in the E. coli proteome following induction and comparison against the uninduced control at 2 distinct time points is achieved using iTRAQ 8-plex reagents. This represents the first study investigating comparative proteomics following the plasmid-based heterologous production of a natural product. The findings contribute to the ongoing discussion concerning the role of microcystin in the toxic cyanobacterial species that produce it, and inform on strategies to increase production efficiency in the E. coli host.