The article introduces a novel method called single-cell structure probing of RNA transcripts (sc-SPORT) to simultaneously determine RNA secondary structure and gene expression at the single-cell level. This method overcomes the limitations of current single-cell RNA sequencing techniques, which cannot directly study RNA structure. Sc-SPORT is applied to human embryonic stem cells (hESCs) and differentiating neurons, revealing that RNA structures are more homogeneous in hESCs compared to neurons, with the highest homogeneity in coding regions. The study also finds extensive heterogeneity in 3' untranslated regions (UTRs) and associates it with specific RNA-binding proteins (RBPs). Overall, RNA structure profiles better discriminate cell type identity and differentiation stage than gene expression profiles alone. The method also identifies a cell-type variable region in 18S ribosomal RNA associated with cell cycle and translation control. The findings highlight the importance of RNA structure in defining cellular identities and suggest that sc-SPORT can provide valuable insights into the dynamics and regulation of RNA structures in individual cells.The article introduces a novel method called single-cell structure probing of RNA transcripts (sc-SPORT) to simultaneously determine RNA secondary structure and gene expression at the single-cell level. This method overcomes the limitations of current single-cell RNA sequencing techniques, which cannot directly study RNA structure. Sc-SPORT is applied to human embryonic stem cells (hESCs) and differentiating neurons, revealing that RNA structures are more homogeneous in hESCs compared to neurons, with the highest homogeneity in coding regions. The study also finds extensive heterogeneity in 3' untranslated regions (UTRs) and associates it with specific RNA-binding proteins (RBPs). Overall, RNA structure profiles better discriminate cell type identity and differentiation stage than gene expression profiles alone. The method also identifies a cell-type variable region in 18S ribosomal RNA associated with cell cycle and translation control. The findings highlight the importance of RNA structure in defining cellular identities and suggest that sc-SPORT can provide valuable insights into the dynamics and regulation of RNA structures in individual cells.