The previous study suggested that spindlin1 is a new tumor related protein which is localized to nuclear and may be involved in regulating cell cycle via TCF-4 pathway. To further study mechanism of spindlin1 function, based on the bioinformatics analysis of spindlin1 structure, A series of spindlin1wild or mutant expression vectors was constructed to determine the key amino acid points for spindin1 localization and function. As previously reported, wild type spindlin1 protein was localized in the nuclei of HeLa cells. Cells transfected with construct either with mutation of Ser14＋Ser84, Ser84＋Ser99 or Ser14＋Ser84＋Ser99 exhibited a cytoplasm spindlin1 expression in a diffused manner, while in those cells transfected with construct with mutation of Ser14，Ser84, Ser99 or Ser14＋Ser 99, the spinlin1 showed a similar subcellular nuclear location as wild type spindlin1. Further TCF-4 reporter assay were performed using these wild type or mutant spindlin1 constructs, and the results indicated that mutation of Ser84 with either Ser14 or Ser99 could abolish the promotion of spindlin1 on TCF-4 reporter activity. All these result suggested that Ser84 of spindlin1, with the cooperation of Ser14 or Ser99 play a key role in it nuclear localization and its function in regulation of TCF-4 pathway.