Batch cultures of Candidatus Pelagibacter ubique were grown under iron-, organosulfur-, and nitrogen-limiting conditions to understand how this ubiquitous marine bacterium responds to and interacts with environments where growth is limited by the availability of these nutrients. Global gene expression was monitored using microarrays and quantitative mass spectrometry to observe both transcriptional and post-transcriptional responses to nutrient limitation. Iron- and nitrogen-limited cultures were characterized by increased transcription and translation of transporters involved in acquisition of the limiting nutrient, whereas organosulfur-limited cultures were not. Methionine synthesis genes downstream of S-adenosyl methionine riboswitches were up-regulated in mRNA and protein during organosulfur-replete stationary phase. Comparative genomics also revealed Ca. Pelagibacter to be the only genus among the free-living Alphaproteobacteria to lack a P[subscript II]-mediated nitrogen regulatory pathway – a pathway which may be complemented in Ca. P. ubique by putative riboswitches and a citric acid cycle able to bypass the glutamate precursor 2-oxoglutarate. Overall, the results of this study provide insight into the regulatory and metabolic processes of this ecologically significant organism, and enable better interpretation of metatranscriptomic and meta-proteomic surveys by identifying sfuC and amtB as likely biomarkers for iron and nitrogen limitation, respectively, in natural Ca. P. ubique populations.