Galectin-3 (gal-3) and its ligands have been implicated in cell transformation and cancer metastasis. Gal-3 protein has been found in uterine epithelial cells adjacent to implanting blastocysts in different cell types. In order to investigate the role of gal-3 in the establishment of human endometrial receptivity, the expression of gal-3 in human endometrial cell line RL95-2 was silenced by RNA interference technology using gal-3 specific small RNA. The expression of gal-3 was detected by the reverse transcriptase-polymerase chain reaction and Western blot analysis. After the suppression of gal-3, cell cycle changes and the expression of integrin β1 were detected by flow cytometry. The adhesive ability of RL95-2 cells was analyzed by the adhesion test. Gal-3 siRNA transfection efficiency reached 70%-90%. The expression of gal-3 mRNA and protein in RL95-2 cells was strongly inhibited by 70%-90% after RNA interference. Inhibition of gal-3 expression decreased S-phase but increased G1 phase cells. Integrin β1 expression was down-regulated, and the adhesive ability of RL95-2 cells to fibronectin (FN) was significantly reduced. Gal-3 may be involved in the establishment of endometrial receptivity by regulating the proliferation and adhesion of endometrial cells. The influence on adhesion may be related with the integrin modulation.