Summary Viral infection of marine phytoplankton releases a variety of dissolved organic matter (DOM). The impact of viral DOM (vDOM) on the uninfected co‐occurring phytoplankton remains largely unknown. Here, we conducted transcriptomic analyses to study the effects of vDOM on the cyanobacterium Prochlorococcus, which is the most abundant photosynthetic organism on Earth. Using Prochlorococcus MIT9313, we showed that its growth was not affected by vDOM, but many tRNAs increased in abundance. We tested tRNA‐gly and found that its abundance increased upon addition of glycine. The decreased transcript abundances of N metabolism genes also suggested that Prochlorococcus responded to organic N compounds in vDOM. Addition of vDOM to Prochlorococcus reduced the maximum photochemical efficiency of photosystem II and CO2 fixation while increasing its respiration rate, consistent with differentially abundant transcripts related to photosynthesis and respiration. One of the highest positive fold‐changes was observed for the 6S RNA, a noncoding RNA functioning as a global transcriptional regulator in bacteria. The high level of 6S RNA might be responsible for some of the observed transcriptional responses. Taken together, our results revealed the transcriptional regulation of Prochlorococcus in response to viral lysis products and suggested its metabolic potential to utilize organic N compounds.