Pseudomonas putida KT2440 is a metabolically versatile soil bacterium useful both as a model biodegradative organism and as a host of catalytic activities of biotechnological interest. In this report, we present the high-resolution transcriptome of P. putida cultured on different carbon sources as revealed by deep sequencing of the corresponding RNA pools. Examination of the data from growth on substrates that are processed through distinct pathways (glucose, fructose, succinate and glycerol) revealed that ≥ 20% of the P. putida genome is differentially expressed depending on the ensuing physiological regime. Changes affected not only metabolic genes but also a suite of global regulators, e.g. the rpoS sigma subunit of RNA polymerase, various cold-shock proteins and the three HU histone-like proteins. Specifically, the genes encoding HU subunit variants hupA, hupB and hupN drastically altered their expression levels (and thus their ability to form heterodimeric combinations) under the diverse growth conditions. Furthermore, we found that two small RNAs, crcZ and crcY, known to inhibit the Crc protein that mediates catabolite repression in P. putida, were both down-regulated by glucose. The raw transcriptomic data generated in this work is made available to the community through the Gene Expression Omnibus database.