BACKGROUND: This work is devoted to the study of the amino acid composition of the fragment of the protein translated from the intron-retaining transcript of the Nxf1 gene. The constitutive protein NXF1 is involved in the process of nuclear-cytoplasmic transport of mRNA. In addition to the constitutive NXF1 protein, there exists an isoform called sNXF1 (“s” for “short”). This isoform is a product of translation of the intron-retaining transcript. There is an early stop codon in the intron-retaining transcript, so the sNXF1 protein is truncated. We have shown that the fragment of this protein corresponding to the beginning of the conserved intron exhibits evolutionary conservation. AIM: This study aimed to analyze the species specificity of the sequence of the C-terminal fragment of the sNXF1 protein translated from the intron. METHODS: The study was performed employed in silico methods, using the sequences of the Nxf1 gene of 33 representatives of the order Rodentia as materials, obtained from publicly available databases and NCBI data. Alignment of the nucleotide sequences was performed using the program MEGA-X, version 10.1.7, using the MUSCLE algorithm (GAP Open-400, 16 iterations). The UPGMA method was used for clustering. Further analysis was carried out using the Unipro UGENE version 36.0. program. Modeling of the protein structure was done using the SWISS-MODEL service with default settings. Phylogenetic trees were created using the Bayes Inference method in the MrBayes program and visualized using FigTree.v1.4.4. Posterior probability percentages were considered reliable if they were >80%–90%. MrBayes parameters were set in the Mesquite program. In our study, model GTR+I+G (General time reversible [GTR] was used; six types of substitution and a combination of the invariable sites model (Proportion of Invariable Sites) with a gamma-model (+I+G, rates=invgamma) were considered. To convert files between formats when performing intermediate operations, the BioEdit v.7.2.5 program was used. RESULTS: The analysis revealed that the C-terminal fragments of the sNXF1 from some representatives of the order Rodentia are of increased length. It was found that the cause of these extended fragments in all cases was a deletion of a cytosine nucleotide. In all identified cases, regardless of the systematic position of the species, the extended fragment contains a sequence of 13 conserved amino acids. CONCLUSION: The obtained results indicate that the analyzed sequence fragment does not influence the conformation of the protein the sNXF1 overall. However, since the fragment has a specific folding pattern, it might determine a set of RNP-complex partners.