Musashi1 (Msi1) is an RNA-binding protein that is involved in cell fate determination. Here, we report the H-1, N-15, and C-13 resonance assignments of Msi1 second RNA-binding domain in free form and in complex with RNA. The assignments can be utilized for NMR structure and dynamics analyses of the Msi1:RNA complex, and moreover, for chemical shift perturbation analyses to evaluate the binding of potential small molecule inhibitors against Msi1:RNA interaction.