Phosphohistidine signaling in mammalian biological systems has not been well characterized. In this study, we investigated the impact of phosphohistidine phosphatase 1 (PHPT1) expression on the hepatic proteome profile in vivo. PHPT1 overexpression was carried out using adenoviral-based transgene delivery for liver-specific overexpression of PHPT1 while a heterozygous knockout was used as a model of PHPT1 deficiency. These two models were characterized using mass spectrometry-based proteomics and label-free relative quantitation. In conjunction with bioinformatic analysis, PHPT1-induced changes in several key proteins and pathways were identified in the liver. Implementation of these models in future studies will lead to far greater insight into the role of phosphohistidine signaling and PHPT1 regulation in mammalian cells.