Objective <p>Ovarian cancer (OC) is a highly aggressive malignancy in females. We aim to investigate the potential gene target and examine its impact on OC.</p> Methods <p>Hub genes were determined using protein–protein interaction networks based on differently expressed genes in GSE12470 and GSE14407 datasets. The impact of FAM64A on the malignant phenotype of OC cells was evaluated by cell counting kit-8, 5-ethynyl-2'-deoxyuridine staining, wound healing, and transwell assays. The epithelial-mesenchymal transition (EMT) process was assessed by determining the protein expression of E-cadherin, N-cadherin, and Vimentin.</p> Results <p>We identified the 18 hub genes of OC with substantial predictive value. FAM64A was selected as a candidate gene. The silencing of FAM64A suppressed the viability (si-NC: 0.78 ± 0.04, 0.95 ± 0.08; si-FAM64A: 0.58 ± 0.05, 0.64 ± 0.11), proliferation (si-NC: 100.00 ± 9.36, 100.00 ± 14.70; si-FAM64A: 34.79 ± 8.88, 44.55 ± 4.91), migration (si-NC: 61.92 ± 8.06, 60.08 ± 5.22; si-FAM64A: 45.88 ± 8.36, 37.78 ± 7.29), and invasion (si-NC: 130.00 ± 10.34, 144.00 ± 13.40; si-FAM64A: 81.00 ± 16.99, 115.60 ± 13.30) of A2780 and SKOV3 cells. FAM64A silencing reduced the EMT in OC cells. The Hippo pathway was identified as the central pathway implicated in the regulatory role of FAM64A in OC. The silencing of FAM64A caused an increase in the protein expression within the Hippo pathway in both A2780 and SKOV3 cells.</p> Conclusion <p>Knockdown of FAM64A emerges as a promising therapeutic target for OC, exerting an inhibitory role in OC by activating the Hippo pathway.</p>