As a new type of post-translational modification (PTM), lysine 2-hydroxyisobutyrylation (K-hib) was firstly identified in histones and functioned as a regulator of transactivation in mammals. However, the role of K-hib proteins remains to be investigated. Here, we firstly identified 10,367 K-hib sites on 2,325 modified proteins in seven patients with pancreatic cancer by applying liquid chromatography with tandem mass spectrometry (LC-MS/MS) qualitative proteomics techniques. Among them, 27 K-hib-modified sites were identified in histones. Bioinformatics analysis revealed that the K-hib-modified proteins were mainly distributed in the cytoplasm and enhanced in metabolic pathways, including glycolysis/gluconeogenesis, the tricarboxylic acid cycle (TCA cycle), and fatty acid degradation. In an overlapping comparison of lysine 2-hydroxyisobutyrylation, succinylation, and acetylation in humans, 105 proteins with 80 sites were modified by all three PTMs, suggesting there may be a complex network among the different modified proteins and sites. Furthermore, MG149, which was identified as a Tip60 inhibitor, significantly decreased the total Khib modification level in pancreatic cancer (PC) and strongly suppressed PC's proliferation, migration, and invasion ability. Overall, our study is the first profiling of lysine 2-hydroxyisobutyrylome and provides a new database for better investigating K-hib in PC.