Tyrosine phosphorylation is emerging as a key post-transcriptional regulator of virulence in a range of bacterial pathogens. However, much is still to be understood, including the specific proteins phosphorylated, the sequence motifs recognised for phosphorylation, and the functional affects these modifications have on pathogenic fitness. In this study, we uncover the diversity of tyrosine phospho-modifications in the human enteropathogen Shigella flexneri. A whole-cell tyrosine phosphoproteome analysis was conducted, identifying 907 unique tyrosine phosphorylation sites on at least 573 proteins. This is the most tyrosine phosphorylated sites identified in a bacterium to date, and is substantially more than levels seen in eukaryotic cells. Phosphorylated proteins included those involved in metabolism homeostasis, cell structure and gene regulation. Importantly, a significant number of proteins associated with virulence were phosphorylated including the Type 3 Secretion System ATPase Spa47 and the master virulence regulator VirB. In order to investigate the functional effects of a number of novel phosphorylation sites, phospho-mimetic and ablative mutations were constructed in both Spa47 and VirB. These mutations disrupted the function of these essential virulence factors, resulting in reduced T3SS effector secretion, modified virulence gene regulation, and decreased virulence by in vitro virulence measures. Together, this work is a further confirmation that tyrosine phosphorylation plays a critical role in the ability of the pathogen to respond to changing environmental conditions, and that the kinases and phosphatases responsible for regulating these modifications may be ideal targets for the development of novel anti-infectives.