A proteomic analysis was performed to estimate the role of BnPHT5;1a in Pi fluctuation. Results showed that Pi deprivation disturbed the physiological processes involved in carbohydrate metabolism, response to stimulus and stress in B. napus, while disruption of BnPHT5;1a genes exacerbated these processes. Besides, the processes of cell redox homeostasis, lipid metabolic and proton transmembrane transport were also unbalanced in BnPHT5;1a DM under the -Pi condition. Noteworthy, disruption of BnPHT5;1a genes severely altered ATP biosynthesis, and proton/inorganic cation transmembrane under normal Pi condition, which might contribute to B. napus growth limitations. Additionally, seven new protein markers of Pi homeostasis were identified in B. napus.