Recently, single-cell metabolomics has been widely used for studying cellular metabolic flow and heterogeneity in biology. Most analytical methods for studying the metabolic flow require sample preparation including tissue isolation, metabolite extraction and purification, whereas in some plant sciences on-site cell-specific analysis is needed because of the limited sap volume from the heterogeneous cells in the living plants. In this view, direct cell metabolic flow analysis combined with 13 C tracer would be promising; however, to our knowledge no attempts have been made with 13 C tracer yet. In this work, growing Egeria densa plants were fed with 13 C to continuously trace carbon and photosynthesis metabolisms in the single leaf cells by using as a single-cell metabolomics technique ‘picolitre pressure-probe electrospray-ionisation mass spectrometry’ (picoPPESI-MS). When the same volume of cell fluids containing chloroplast(s) were collected and the metabolites in the fluids were assayed, numerous metabolites involved in carbon metabolism have been detected. When 13 C/12 C isotope ratio of each metabolite was determined with an Orbitrap mass analyser, the kinetics has revealed that 13 C fed as carbon dioxide was preferentially taken into the Calvin-Benson cycle, and subsequently photorespiration, followed by TCA cycles and glycolysis in the order in the same single cells. And hence, we conclude that picoPPESI-MS is capable for directly determining the dynamics of metabolic flow at the inter-organelle resolution in the target cells and this analytical method combined with 13 C tracer might be useful for various research subjects in biology.