The present study aimed to investigate the potential effect of fibronectin (FN) in adenomyosis progression. Small guide RNAs were designed to knock down FN expression in Ishikawa cells. The impact of FN on the proliferation, apoptosis, migration, and invasion of the cells was assessed. Cell proliferation was detected using a Celigo Imaging Cytometer system; apoptosis was quantified by flow cytometry; and cell migration and invasion were investigated via transwell assays. Cell proliferation was markedly suppressed in the FN knockout (KO) group compared with the control group, while apoptosis significantly increased. The levels of cell migration and invasion in the KO group were significantly decreased compared with the control group. Our study revealed that downregulation of FN expression is likely to restrain cell proliferation, migration, and invasion in endometrial cells in adenomyosis.