High-grade serous ovarian cancer is an aggressive form of epithelial ovarian cancer (EOC), and accounts for the majority of deaths due to EOC. The critical cellular processes and underlying molecular mechanisms that define this malignancy remain poorly understood. Using a syngeneic murine model, we investigated the changes that accompanied the progression to increased aggressiveness induced by in vivo passage of mouse EOC cells. We found that enhanced anoikis resistance was a key cellular process associated with greater aggressiveness and tumorigenicity in vivo. Biochemical studies revealed that the enhanced anoikis resistance was associated with the activation of the Src/Akt/Erk signaling pathway. A higher rate of metabolism and autophagy were also associated with increased anoikis resistance. Blocking these pathways with specific inhibitors and/or genetic modifications significantly increased anoikis in vitro and inhibited tumor development in vivo. In addition, we demonstrated that similar signaling pathways were also involved in a human EOC cell line model. Collectively, our data suggest that anoikis resistance represents a critical and a distinguishing feature underlying the aggressiveness of ovarian cancer cells.