Deep-water nutrient injection in the North Pacific Subtropical Gyre provides an aperiodic yet significant source of inorganic nutrients to the nutrient-limiting surface waters, and has been implicated in phytoplankton bloom formation. Here we examined short-term transcriptional responses of surface water picoplankton assemblages in a deep-sea water (DSW) mixing experiment. Both flow cytometric and transcriptomic analysis indicated stimulation of an Alteromonas-like population in the DSW-amended treatment after 12 h, relative to the control. Among the highly expressed alteromonad transcripts in DSW-treated samples, those encoding genes associated with chemotaxis, cell motility and carbon metabolism were most highly represented, relative to the control. Similarly, Prochlorococcus showed significantly higher levels of transcripts associated with carbon fixation and photosynthesis, as well as slightly increased cell density, relative to the control. Although other microbial taxa did not exhibit enhanced growth, DSW-stimulated changes in their genome-wide transcriptional profiles were still readily detectable. Cell-associated cyanophage DNA and cDNA profiles suggested DSW stimulation of phage-mediated cell lysis, in previously infected cells. Comparison of the DSW-responsive Alteromonas populations, to previously reported dissolved organic matter (DOM)-responding alteromonads (McCarren et al., 2010), revealed differential transcript abundances, predominantly among genes encoding mobile elements and phage-related genes. Transcript representation in other metabolic pathways differed significantly between Alteromonas populations in the two different treatments, suggesting perturbation-specific metabolic responses to DSW and DOM. In total, the results provide new insight into short-term responses of picoplankton to DSW mixing, which occur prior to the more well-studied, longer-term growth responses of larger phytoplankton species.