Modifications are present on many classes of RNA, including tRNA, rRNA, and mRNA. These modifications modulate diverse biological processes such as genetic recoding and mRNA export and folding. In addition, modifications can be introduced to RNA molecules using chemical probing strategies that reveal RNA structure and dynamics. Many methods exist to detect RNA modifications by short-read sequencing; however, limitations on read length inherent to short-read-based methods dissociate modifications from their native context, preventing single-molecule modification analysis. Here, we demonstrate direct RNA nanopore sequencing to detect endogenous and exogenous RNA modifications on long RNAs at the single-molecule level. We detect endogenous 2′-O-methyl and base modifications across E. coli and S. cerevisiae ribosomal RNAs as shifts in current signal and dwell times distally through interactions with the helicase motor protein. We further use the 2′-hydroxyl reactive SHAPE reagent acetylimidazole to probe RNA structure at the single-molecule level with readout by direct nanopore sequencing. Display omitted Nanopore sequencing detects endogenous RNA modifications at a single-molecule level2′-O-methyl (Nm) and pseudouridine (Ψ) alter nanopore translocation kineticsAcIm is a small-adduct-generating SHAPE-MaP reagentAcIm enables full-length single-molecule structural profiling on RNA Stephenson et al. employ direct RNA nanopore sequencing to detect endogenous and exogenous modifications on single RNA molecules. The authors demonstrate detection of endogenous 2′-O-methylation (Nm) on native ribosomal RNAs, confirming known modification patterns. They describe the development of nanoSHAPE, a method that involves exogenously labeling RNA with a small-adduct-generating chemical probe that can reveal RNA structure using long-read sequencing.