AimsMechanisms for carbon fixation via photosynthesis in the diatom Phaeodactylum tricornutum Bohlin were studied recently but there remains a long-standing debate concerning the occurrence of C-4 photosynthesis in this species. A thorough investigation of carbon metabolism and the evidence for C-4 photosynthesis based on organelle partitioning was needed. Methods and ResultsIn this study, we identified the flux ratios between C-3 and C-4 compounds in P.tricornutum using C-13-labelling metabolic flux ratio analysis, and stained cells with various cell-permeant fluorescent probes to investigate the likely organelle partitioning required for single-cell C-4 photosynthesis. Metabolic flux ratio analysis indicated the C-3/C-4 exchange ratios were high. Cell staining indicated organelle partitioning required for single-cell C-4 photosynthesis might exist in P.tricornutum. ConclusionThe results of C-13-labelling metabolic flux ratio analysis and cell staining suggest single-cell C-4 photosynthesis exists in P.tricornutum. Significance and Impact of the StudyThis study provides insights into photosynthesis patterns of P.tricornutum and the evidence for C-4 photosynthesis based on C-13-labelling metabolic flux ratio analysis and organelle partitioning.