Background: As a potent mediator of hypothermic neuroprotection, the cold-inducible protein RBM3 is characterized with one RNA-recognition motifs (RRM) and one arginine-glycine-rich (RGG) domain. It is known that these conserved domains are required for nuclear localization in some RNA-binding proteins. However, little is known about the actual role of RRM and RGG domains in subcellular localization of RBM3. Methods: To clarify it, various mutants of human Rbm3 gene were constructed. Plasmids were transfected into cells and the localization of RBM3 protein and its varias mutants in cells and role in neuroprotection. Results: In human neuroblastoma SH-SY5Y cells, either a truncation of RRM domain (aa 1–86) or RGG domain (aa 87–157) led to an obvious cytoplasmic distribution, compared to a predominant nuclear localization of whole RBM3 protein (aa 1–157). In contrast, mutants in several potential phosphorylated sites of RBM3, including Ser102, Tyr129, Ser147, and Tyr155, did not alter the nuclear localization of RBM3. Similarly, mutants in two Di-RGG motif sites also did not affect the subcellular distribution of RBM3. Lastly, the role of Di-RGG motif in RGG domains was further investigated. The mutant of double arginines in either Di-RGG motif-1 (Arg87/90) or -2 (Arg99/105) exhibited a higher cytoplasmic localization, indicating that both Di-RGG motifs are required for nucleic localization of RBM3. Conclusions: Our data suggest that RRM and RGG domains are both required for the nuclear localization of RBM3, with two Di-RGG domain being crucial for nucleocytoplasmic shuttling of RBM3.