The classical nuclear import pathway in eukaryotic cells is mediated by Importin (IMP) subunits alpha (IMPα) and beta (IMPβ1). IMPα functions as an adaptor protein that recognizes and binds nuclear localization signals (NLSs) on cargo proteins, while IMPβ1 mediates the translocation of the import complex through the nuclear pore complex. NLS recognition by IMPα is a critical step in nucleocytoplasmic transport, regulating signal transduction and gene expression. IMPα from the causative agent of toxoplasmosis, Toxoplasma gondii (TgIMPα) is an essential gene and validated drug target, but its NLS recognition mechanisms remain poorly understood. To investigate the NLS binding properties of bacterially expressed TgIMPα, we designed NLS peptides and conducted binding assays using AlphaScreen technology. The viral canonical NLS derived from SV40 Large T-antigen (SV40 T-ag NLS), exhibited low-nanomolar binding affinity to TgIMPα, comparable to mouse IMPα. In contrast, a T. gondii-specific NLS from TgGCN5 displayed strong and selective binding to TgIMPα with negligible affinity for the mammalian homolog. Mutant NLS peptides were used to dissect the role of key basic residues in complex formation. To elucidate the structural basis of these interactions, an ab initio model of TgIMPα was generated, and molecular dynamics simulations were performed to compare the NLS-binding pockets of TgIMPα with mammalian IMPα. Parallel simulations with TgGCN5 NLS revealed conserved and parasite-specific features governing TgIMPα selectivity. This study provides insights into the molecular distinctions between parasite and host IMPα, offering a foundation for the development of targeted drug design or peptidomimetic strategies against T. gondii.