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Abstract: Objective To detect the expression of non-secretory CXCL16 and its impact on malignant biological behaviors in breast cancer cell lines. Methods RT-PCR was carried out to examine the expression of CXCL16 mRNA in breast cancer cell lines with different aggressiveness SK-BR-3, MCF-7, MDA-MB-231, MDA-MB-435S and human mammary normal epithelial celll MCF-10A. The eukaryotic expression plasmid of CXCL16 was transfected into MDA-MB-231 cells and overexpression of CXCL16 was confirmed by Real time PCR and Western blot. Boyden Chamber assay was used to determine cell migration and invasion, while MTT assay was performed to determine cell proliferation. Results Among four breast cancer cell lines, CXCL16 mRNA was highly expressed in MCF-7, lowly expressed in MDA-MB-231, while MCF-10A faintly expressed CXCL16. Overexpression of CXCL16 led to a decrease in cell migration, invasion but not proliferation in MDA-MB-231 cells. Conclusion Expression of non-secretory CXCL16 is associated with aggressiveness of breast cancer cell lines, and CXCL16 expression inhibits cell migration and invasion in vitro.