Posttranslational modifications alter the biophysical properties of proteins and thereby influence cellular physiology. One emerging manner by which such modifications regulate protein functions is through their ability to perturb protein stability. Despite the increasing interest in this phenomenon, there are few methods that enable global interrogation of the biophysical effects of posttranslational modifications on the proteome. Here, we describe an unbiased proteome-wide approach to explore the influence of protein modifications on the thermodynamic stability of thousands of proteins in parallel. We apply this profiling strategy to study the effects of O-linked N-acetylglucosamine (O-GlcNAc), an abundant modification found on hundreds of proteins in mammals that has been shown in select cases to stabilize proteins. Using this thermal proteomic profiling strategy, we identify a set of 72 proteins displaying O-GlcNAc-dependent thermostability and validate this approach using orthogonal methods targeting specific proteins. These collective observations reveal that the majority of proteins influenced by O-GlcNAc are, surprisingly, destabilized by O-GlcNAc and cluster into distinct macromolecular complexes. These results establish O-GlcNAc as a bidirectional regulator of protein stability and provide a blueprint for exploring the impact of any protein modification on the meltome of, in principle, any organism.