Abstract Mammalian genome is hierarchically organized by CTCF and cohesin through loop extrusion mechanism to facilitate the organization of topologically associating domains (TADs). Mounting evidence suggests additional factors/mechanisms exist to orchestrate TAD formation and maintenance. In this study, we investigate the potential role of RNA-binding proteins (RBPs) in TAD organization. By integrated analyses of global RBP binding and 3D genome mapping profiles from both K562 and HepG2 cells, our study unveils the prevalent enrichment of RBPs on TAD boundaries and define boundary-associated RBPs (baRBPs). We found that baRBP binding is correlated with enhanced TAD insulation strength and in a CTCF-independent manner. Moreover, baRBP binding is associated with nascent promoter transcription. Additional experimental testing was performed using RBFox2 as a paradigm. Knockdown of RBFox2 in K562 cells causes mild TAD reorganization. Moreover, RBFox2 enrichment on TAD boundaries is a conserved phenomenon in C2C12 myoblast (MB) cells. RBFox2 is downregulated and its bound boundaries are remodeled during MB differentiation into myotubes. Finally, transcriptional inhibition indeed decreases RBFox2 binding and disrupts TAD boundary insulation. Altogether, our findings demonstrate that RBPs can play an active role in modulating TAD organization through co-transcriptional association and synergistic actions with nascent promoter transcripts.