A serum inhibited gene Si1 (GenBank acession number: AY050169) was previously cloned and identified by differential expression of genes in U251 cells. For the further study of biological function of Si1, prediction procedure was performed to predict its subcelluar-localization. Relative experiments were carried out at the same time. The expression of EGFP/Si1 recombinant in HeLa cells showed Si1 protein located in nuclear which corroborated the prediction results of PsortⅡ, Proloc, Cello version2, Subnuclear compartments prediction system, NUCLEO and NUCPRED. According to the PredictNLS prediction, twelve different fragments of EGFP/Si1 recombinants were constructed to identify precise NLS regulation sequence. Findings proved that the real NLS regulation sequence was not the same as the software predicted(1 206 bp～1 239 bp on Si1 ORF), but located on 1 395 bp～1 594 bp of Si1. A tumor relatived mutation/EGFP recombinant localization result showed though the mutation site (1 639 bp on Si1 ORF) does not located in NLS regulation sequence, it did affect wildtype Si1 protein divert to nuclear and may affect its natural function in cell, perhaps it is the main reason for highly mutation rate of Si1 in tumor.