Nucleoporin 98 (NUP98) and nucleoporin 96 (NUP96) are essential components of the nuclear pore complex (NPC) in eukaryote cells. However, there is a lack of available information about complete Rehmannia glutinosa NUP98-96 (rgNUP98-96) sequences. Here, the full-length cDNA sequence of rgNUP96-98 was isolated from R. glutinosa using rapid amplification of cDNA ends (RACE) technology, based on a cloned cDNA sequence (GenBank accession No. JZ483329). The identified rgNUP98-96 was 3476 bp, and it encoded a 1041-amino acid peptide. The BLAST search analysis of rgNUP98-96 showed an intermediate degree of similarity (60-79%) to the NUP98-96 protein sequences of 34 other plants, including the dicotyledons Erythranthe guttata, Genlisea aurea, Coffea canephora, Nicotiana benthamiana, Solanum lycopersicum, and Solanum tuberosum. The phylogenetic analysis of NUP96-98 sequences indicated that R. glutinosa and E. guttata sequences shared the closest homology. The calculated molecular mass and predicted isolectric point of the complex protein were 117.6 kDa and 4.99, respectively. The secondary and three-dimensional structure studies illustrated that the rgNUP96-98 protein folded into a channel motif comprised of 34 alpha-helices, nine beta-strands, and several long loops. Using quantitative real-time PCR, the spatio-temporal expression patterns of rgNUP98-96 were analyzed in R. glutinosa, and the results indicated that rgNUP98-96 was highly expressed at the early stage of R. glutinosa tuberous root expansion, which is associated with a higher expression pattern in roots. The study provides a valuable foundation for further investigation of rgNUP96-98 molecular functions in R. glutinosa.