Objective To examine the mechanism of the eye-open at birth (EOB) phenotype in B6-Co mice at birth. Methods C57BL/ 6 (B6) mice were bred at a ratio of male to female mice of 2 ∶1 to obtain pregnant mice. B6-Co mice were bred by B6 (male) mice with B6-Co (female) mice at a ratio of 1 ∶2 or B6 (female) mice with B6-Co (male) mice at a ratio of 2 ∶ 1 hybridization to obtain pregnant mice. We selected 18. 5-day embryos of pregnant mice and then collected embryonic eyelid tissue to culture mouse eyelid fibroblasts. We used immunofluorescence and hematoxylin and eosin staining to identify primary cells and observe morphology of fibroblasts. The difference in growth curves between two cell types was observed using the direct counting method with a hemocytometer. Real-time PCR and Western blot were used to detect HSP70 expression in the two cell types. Lipofectamin2000 was used to transfect the fibroblasts.The HSP70 gene mRNA and protein expression levels were detected by Real-Time PCR and Western blot to determine the interference efficiency. The migration ability of fibroblasts was detected by Transwell experiment. Results The growth curve of eyelid fibroblasts showed that B6-Co mouse eyelid fibroblasts proliferated more slowly than B6 mouse eyelid fibroblasts ( P< 0. 01). However, HSP70 expression levels in B6-Co mouse fibroblasts were significantly lower than those in B6 mouse fibroblasts. After the fibroblasts were transfected with HSP70 siRNA interference vector, the expression levels of HSP70 gene on mRNA and protein were effectively inhibited. Among them, siRNA-HSP70 - 3 has the strongest interference efficiency, and the interference efficiency was up to 70% (P< 0. 05). The migration abilities of fibroblasts in the siRNA- HSP70 group were significantly lower (P< 0. 05) than those in the siRNA -NC group. Conclusions Downregulation of HSP70 gene and protein expression affects the growth curve of fibroblasts in B6-Co mice and may be involved in regulation of embryonic development of fibroblasts. The constructed siRNA-HSP70- 3 interference vector can effectively inhibit the expression of target gene HSP70 on fibroblasts, and can significantly inhibit the cell migration. This could be one of the important reasons for the EOB phenotype of B6-Co mice.