The ability to utilize dissolved organic phosphorus (DOP) gives phytoplankton competitive advantages in P-limited environments. Our previous research indicates that the diatom Phaeodactylum tricornutum could grow on glyphosate, a DOP with carbon-phosphorus (C-P) bond and an herbicide, as sole P source. However, direct evidence and mechanism of glyphosate utilization are still lacking. In this study, using physiological and isotopic analysis, combined with transcriptomic profiling, we demonstrated the uptake of glyphosate by P. tricornutum and revealed the candidate responsible genes. Our data showed a low efficiency of glyphosate utilization by P. tricornutum, suggesting that glyphosate utilization costs energy and that the alga possessed an herbicide-resistant type of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase. Compared to the P-limited cultures, the glyphosate-grown P. tricornutum cells up-regulated genes involved in DNA replication, cell growth, transcription, translation, carbon metabolism, and many genes encoding antioxidants. Additionally, cellular C and silicon (Si) increased remarkably while cellular nitrogen (N) declined in the glyphosate-grown P. tricornutum, leading to higher Si:C and Si:N ratios, which corresponded to the up-regulation of genes involved in the C metabolism and Si uptake and the down-regulation of those encoding N uptake. This has the potential to enhance C and Si export to the deep sea when P is limited but phosphonate is available. In sum, our study documented how P. tricornutum could utilize the herbicide glyphosate as P nutrient and how glyphosate utilization may affect the element content and stoichiometry in this diatom, which have important ecological implications in the future ocean.IMPORTANCEGlyphosate is the most widely used herbicide in the world and could be utilized as phosphorus (P) source by some bacteria. Our study first revealed that glyphosate could be transported into Phaeodactylum tricornutum cells for utilization and identified putative genes responsible for glyphosate uptake. This uncovers an alternative strategy of phytoplankton to cope with P deficiency considering phosphonate accounts for about 25% of the total dissolved organic phosphorus (DOP) in the ocean. Additionally, accumulation of carbon (C) and silicon (Si), as well as elevation of Si:C ratio in P. tricornutum cells when grown on glyphosate indicates glyphosate as the source of P nutrient has the potential to result in more C and Si export into the deep ocean. This, along with the differential ability to utilize glyphosate among different species, glyphosate supply in dissolved inorganic phosphorus (DIP)-depleted ecosystems may cause changes in phytoplankton community structure. These insights have implications in evaluating the effects of human activities (use of Roundup) and climate change (potentially reducing DIP supply in sunlit layer) on phytoplankton in the future ocean.