While protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well-established as a key regulatory posttranslational modification in eukaryotes, little is known about its extent and function in prokaryotes. Although protein kinases and phosphatases have been predicted and identified in a variety of bacterial species, classical biochemical approaches have so far revealed only few substrate proteins, and even less phosphorylation sites. Bacillus subtilis is a model Gram-positive bacterium in which 2D electrophoresis-based studies suggest that the Ser/Thr/Tyr phosphorylation should be present on more than hundred proteins. However, so far only 16 phosphorylation sites on 8 of its proteins have been determined, mostly in in-vitro studies. Here we performed a global, gel-free and site-specific analysis of the B. subtilis phosphoproteome using high accuracy mass spectrometry in combination with biochemical enrichment of phosphopeptides from digested cell lysates. We identified 103 unique phosphopeptides from 78 B. subtilis proteins and determined 78 phosphorylation sites; 54 on serine, 16 on threonine and 8 on tyrosine. Detected phosphoproteins are involved in a wide variety of metabolic processes but are enriched in carbohydrate metabolism. We report phosphorylation sites on almost all glycolytic and TCA cycle enzymes, several kinases and members of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). This significantly enlarged number of bacterial proteins known to be phosphorylated on Ser/Thr/Tyr residues strongly supports the emerging view that protein phosphorylation is a general and fundamental regulatory process, not restricted only to eukaryotes, and opens the way for its detailed functional analysis in bacteria.