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Protein post-translational modification (PTM) is a reversible process, which can dynamically regulate the metabolic state of cells through regulation of protein structure, activity, localization or protein–protein interactions. Actinomycetes are present in the soil, air and water, and their life cycle is strongly determined by environmental conditions. The complexity of variable environments urges Actinomycetes to respond quickly to external stimuli. In recent years, advances in identification and quantification of PTMs have led researchers to deepen their understanding of the functions of PTMs in physiology and metabolism, including vegetative growth, sporulation, metabolite synthesis and infectivity. On the other hand, most donor groups for PTMs come from various metabolites, suggesting a complex association network between metabolic states, PTMs and signaling pathways. Here, we review the mechanisms and functions of PTMs identified in Actinomycetes, focusing on phosphorylation, acylation and protein degradation in an attempt to summarize the recent progress of research on PTMs and their important role in bacterial cellular processes.