The serine/arginine-rich (SR) proteins are one type of major actors in regulation of pre-mRNA splicing. Their functions are closely related to the intracellular spatial organization. The RS domain and phosphorylation status of SR proteins are two critical factors in determining the subcellular distribution. Mammalian Transformer-2β (Tra2β) protein, a member of SR proteins, is known to play multiple important roles in development and diseases. In the present study, we characterized the subcellular and subnuclear localization of Tra2β protein and its related mechanisms. The results demonstrated that in the brain the nuclear and cytoplasmic localization of Tra2β were correlated with its phosphorylation status. Using deletional mutation analysis, we showed that the nuclear localization of Tra2β was determined by multiple nuclear localization signals (NLSs) in the RS domains. The point-mutation analysis disclosed that phosphorylation of serine residues in the NLSs inhibited the function of NLS in directing Tra2β to the nucleus. In addition, we identified at least two nuclear speckle localization signals within the RS1 domain, but not in the RS2 domain. The nuclear speckle localization signals determined the localization of RS1 domain-contained proteins to the nuclear speckle. The function of the signals did not depend on the presence of serine residues. The results provide new insight into the mechanisms by which the subcellular and subnuclear localization of Tra2β proteins are regulated.