A new method, sc-SPORT, enables the simultaneous determination of RNA secondary structure and abundance at single-cell resolution. This method was applied to human embryonic stem cells (hESCs) and differentiating neurons, revealing that RNA structures are more homogeneous in hESCs compared to neurons, with the greatest homogeneity in coding regions. RNA structure profiles better discriminate cell type identity and differentiation stage than gene expression profiles alone. The study also identified a cell-type variable region of 18S ribosomal RNA associated with cell cycle and translation control. RNA structure heterogeneity is influenced by RNA-binding proteins (RBPs), with 3' untranslated regions (UTRs) showing more heterogeneity. Structural heterogeneity can inform RBP binding and gene regulation, with 3' UTRs of 18S rRNA showing a region associated with translation. Single-cell RNA structure probing revealed that structurally homogeneous transcripts are more abundant and associated with ribosomal assembly, rRNA processing, and translation. Structurally heterogeneous transcripts are associated with mRNA stability, RNA localization, and alternative splicing. The study also showed that structural heterogeneity can help separate cell populations, with RPL41 showing structural changes during neuronal differentiation. sc-SPORT provides a powerful tool for understanding RNA structure-function relationships at single-cell resolution, revealing dynamics and regulation in small cellular populations. The method has potential applications in clustering cells and predicting developmental trajectories, enhancing our understanding of structure-phenotype relationships in diverse biological systems. The study highlights the importance of RNA structure in gene regulation and cellular identity, and the need for further research to fully understand the role of RNA structure in cellular processes.A new method, sc-SPORT, enables the simultaneous determination of RNA secondary structure and abundance at single-cell resolution. This method was applied to human embryonic stem cells (hESCs) and differentiating neurons, revealing that RNA structures are more homogeneous in hESCs compared to neurons, with the greatest homogeneity in coding regions. RNA structure profiles better discriminate cell type identity and differentiation stage than gene expression profiles alone. The study also identified a cell-type variable region of 18S ribosomal RNA associated with cell cycle and translation control. RNA structure heterogeneity is influenced by RNA-binding proteins (RBPs), with 3' untranslated regions (UTRs) showing more heterogeneity. Structural heterogeneity can inform RBP binding and gene regulation, with 3' UTRs of 18S rRNA showing a region associated with translation. Single-cell RNA structure probing revealed that structurally homogeneous transcripts are more abundant and associated with ribosomal assembly, rRNA processing, and translation. Structurally heterogeneous transcripts are associated with mRNA stability, RNA localization, and alternative splicing. The study also showed that structural heterogeneity can help separate cell populations, with RPL41 showing structural changes during neuronal differentiation. sc-SPORT provides a powerful tool for understanding RNA structure-function relationships at single-cell resolution, revealing dynamics and regulation in small cellular populations. The method has potential applications in clustering cells and predicting developmental trajectories, enhancing our understanding of structure-phenotype relationships in diverse biological systems. The study highlights the importance of RNA structure in gene regulation and cellular identity, and the need for further research to fully understand the role of RNA structure in cellular processes.