Abstract: Coral reefs, often referred to as the ‘tropical rainforests of the sea’, are among the most ecologically diverse and productive marine ecosystems on Earth. However, climate change—especially global ocean warming—has triggered widespread coral bleaching events, posing a severe threat to reef biodiversity, structure, and their function. Central to coral resilience is the symbiotic relationship with Symbiodiniaceae, a family of photosynthetic dinoflagellates that provide essential nutrients to the coral host. The environmental adaptability of this symbiosis is strongly influenced by the physiological and metabolic traits of the symbionts. While extensive research has characterized Symbiodiniaceae diversity and abundance at the population level, relatively little is known about the metabolic and functional heterogeneity that exists at the level of individual cells—particularly across phylogenetically distinct clades. To address this gap, a custom-built induced nano-electrospray ionization (Induced nanoESI) single-cell mass spectrometry platform was developed and applied. This platform enabled high-sensitivity, label-free profiling of intracellular metabolites from individual Symbiodiniaceae cells, facilitating a more detailed understanding of their functional and metabolic diversity. This platform was applied to characterize the metabolic profiles of three ecologically divergent Symbiodiniaceae clades, including the light-tolerant Symbiodinium (Clade A), the broadly distributed Cladocopium (Clade C), and the thermotolerant Durusdinium (Clade D). The method demonstrated good reproducibililty and sensitivity in single-cell metabolomics. Clear metabolic differentiation among the three clades was observed using multivariate statistical analyses, including principal coordinate analysis (PCoA) and orthogonal partial least squares discriminant analysis (OPLS-DA). A total of 14 differential metabolites are identified through high-resolution mass spectrometry (HRMS) and further confirmed by liquid chromatography coupled to hybrid high-resolution mass spectrometry (LC-HRMS/MS) spectral matching. Among these, leucine is found to be significantly depleted in Clade A relative to Clades C and D; valine is highly enriched in Clade C; and dimethylsulfoniopropionate (DMSP), a well-known osmoprotectant and antioxidant, is specifically accumulated in Clade D. These results suggest distinct clade-specific metabolic strategies. It was proposed that leucine biosynthesis is downregulated by Clade A to reduce acetyl-CoA accumulation, thereby promoting reduced nicotinamide adenine dinucleotide (NADH) regeneration and enhancing cyclic electron flow under high-light stress. Clade C's elevated valine levels may aid in host nutritional support and symbiotic stability. Clade D's high DMSP production likely contributes to oxidative stress resistance and thermal tolerance. In conclusion, this study highlights the power of single-cell metabolomics in uncovering functional diversity within symbiodiniaceae. New molecular insights into coral-symbiont interactions are provided by our findings, and a foundation is established for understanding how clade-specific metabolic adaptations contribute to coral reef resilience in a changing climate.