If left untreated, some cervical high-grade squamous intraepithelial lesions will progress to invasive squamous cell carcinoma (SCC), but the molecular events conferring invasive potential remain poorly defined. In prior work, we identified 48 genes that were down-regulated in SCCs compared with high-grade squamous intraepithelial lesions and normal squamous epithelia. In this study, a functional screening strategy was used to identify which of these genes regulate cervical cancer cell invasion. Two independent squamous epithelial cell lines were transduced with a library of short hairpin RNAs targeting the differentially expressed genes and tested for invasion of the chick chorioallantoic membrane. PCR was used to recover specific short hairpin RNAs from cells that invaded the chorioallantoic membrane. Constructs targeting estrogen receptor 1 (ESR1) were highly enriched in the invasive cells. The short hairpin RNA-mediated inhibition of ESR1 in SCC- and precancer-derived cell lines increased invasiveness in both in vivo and in vitro assays. Conversely, restoration of ESR1 expression in ESR1-negative cervical cancer cells reduced cell invasiveness. Loss of ESR1 expression was found to accompany cervical cancer progression in an analysis of primary normal cervix, low grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and SCC specimens. Molecular mechanisms underlying down-regulation of ESR1 in invasive cervical carcinomas appear to be complex and likely heterogeneous. Our findings indicate that loss of ESR1 has a major role in mediating cervical cancer invasion and progression.