Microcystis blooms are the most widely distributed and frequently occurring cyanobacterial blooms in freshwater. Reducing phosphorus is suggested to be effective in mitigating cyanobacterial blooms, while the underlying molecular mechanisms are yet to be elucidated. In the present study, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics was employed to study the effects of phosphorus depletion on Microcystis aeruginosa FACHB-905. The production of microcystins (MCs), a severe hazard of Microcystis blooms, was also analyzed. In total, 230 proteins were found to be differentially abundant, with 136 downregulated proteins. The results revealed that, upon phosphorus limitation stress, Microcystis aeruginosa FACHB-905 raised the availability of phosphorus primarily by upregulating the expression of orthophosphate transport system proteins, with no alkaline phosphatase producing ability. Phosphorus depletion remarkably inhibited cell growth and the primary metabolic processes of Microcystis, including transcription, translation and photosynthesis, with structures of photosystems remaining intact. Moreover, expression of nitrogen assimilation proteins was downregulated, while proteins involved in carbon catabolism were significantly upregulated, which was considered beneficial for the intracellular balance among carbon, nitrogen and phosphorus. The expression of MC synthetase was not significantly different upon phosphorus depletion, while MC content was significantly suppressed. It is assumed that phosphorus depletion indirectly regulates the production of MC by the inhibition of metabolic processes and energy production. These results contribute to further understanding of the influence mechanisms of phosphorus depletion on both biological processes and MC production in Microcystis cells.