RBM24 is a critical post-translational regulator involved in the regulation of RNA stability, RNA splicing, cap-dependent and cap-independent translation. Previously, we demonstrated that RBM24 prevents 80S ribosome assembly. However, the detailed mechanisms remain unknown. Here, we identified RPL3, a ribosomal 60S subunit protein, as an interaction partner of RBM24. Pulldown assays with truncated RPL3 indicated that RBM24 binds both exposed and 28S-embeded region of RPL3. Ribosome affinity capture assay revealed that RBM24 enhances the interaction between RPL3 and eIF6, a translation initiation factor that prevents the association of the 40S and 60S ribosomal subunits. Further analysis revealed that RBM24 did not interact with eIF6. Moreover, RBM24 blocked the binding of eIF6 to 28S-embeded region of RPL3, but did not affect the binding of eIF6 to reported eIF6-contacting region of RPL3 in 60S subunit. Peptide competitor derived from eIF6-contacting region of RPL3 rescue RBM24 mediated translation inhibition in vitro. In conclusion, the present study demonstrated that RBM24 binds RPL3 and facilitates eIF6-RPL3 interaction, and elucidated a probably explanation to the inhibition of 80S ribosome assembly by RBM24 observed in previous studies.