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Abstract Iron is an essential nutrient for all microorganisms of the marine environment. Iron limitation of primary production has been well documented across a significant portion of the global surface ocean, but much less is known regarding the potential for iron limitation of the marine heterotrophic microbial community. In this work, we characterize the transcriptomic response of the heterotrophic bacterial community to iron additions in the California Current System, an eastern boundary upwelling system, to detect in situ iron stress of heterotrophic bacteria. Changes in gene expression in response to iron availability by heterotrophic bacteria were detected under conditions of high productivity when carbon limitation was relieved but when iron availability remained low. The ratio of particulate organic carbon to dissolved iron emerged as a biogeochemical proxy for iron limitation of heterotrophic bacteria in this system. Iron stress was characterized by high expression levels of iron transport pathways and decreased expression of iron-containing enzymes involved in carbon metabolism, where a majority of the heterotrophic bacterial iron requirement resides. Expression of iron stress biomarkers, as identified in the iron-addition experiments, was also detected in  situ. These results suggest iron availability will impact the processing of organic matter by heterotrophic bacteria with potential consequences for the marine biological carbon pump.