Prokaryotic ubiquitin-like protein (Pup) is the first identified prokaryotic protein that is functionally analogous to ubiquitin. Recent studies have shed light on the Pup activation and conjugation to target proteins to be a signal for the selective degradation proteins in Mycobacterium tuberculosis (Mtb). By covalently conjugating the Pup, pupylation functions as a critical post-translational modification (PTM) conserved in actinomycetes. Detecting pupylation sites is crucial and fundamental for understanding the molecular mechanisms of Pup. Yet comparative studies with other PTM suggest that the development of accurate and complete repertories of pupylation is still in its early stages. Unbiased screening for pupylation sites by experimental methods is time consuming and expensive; in silico prediction can provide highly potential candidates and reduce the number of potential candidates that require further in vivo or in vitro confirmation. Here, we present an effective classifier of PupPred for predicting pupylation sites, which shows better performance than existing classifiers. Importantly, this work not only investigates the sequential, structural and evolutionary hallmarks around pupylation sites but also compares the differences of pupylation and ubiquitylation from the environmental, conservative and functional characterization of substrates. These prediction and analysis results may be helpful for further experimental investigation of degradation proteins in prokaryotes. Finally, the PupPred server is available at http://bioinfo.ncu.edu.cn/PupPred.aspx.