Gastric cancer (GC) is the most common malignant tumor of the digestive tract. However, the molecular pathogenesis is not well understood. Through bioinformatic analysis and analyzing clinical tissue samples, we found that E2F1 and E2F7 as well as their potential downstream target MYBL2 were all upregulated in GC tissues and that their expressions correlated with patient prognosis. While knockdown of E2F1 or MYBL2 inhibited cell proliferation and promoted apoptosis, knockdown of E2F7 promoted cell proliferation but had no effects on apoptosis. Chromatin immunoprecipitation and dual luciferase reporter assays demonstrated that MYBL2 was transcriptionally activated and repressed by E2F1 and E2F7, respectively. Importantly, in vitro and ex vivo experiments demonstrated that the effects of E2F1 and E2F7 on GC cell proliferation were significantly attenuated by reversely modulating MYBL2 expression, indicating that MYBL2 is a direct and functionally relevant target of E2F1 and E2F7 in GC cells. Furthermore, the effects of E2F1 and E2F7 on GC cell proliferation through transcriptional regulation of MYBL2 can be mediated by the PI3K/AKT signaling pathway. Interestingly, we found differential nucleocytoplasmic distribution of E2F7 in GC cells with functional relevance. Taken together, our data suggest that targeted therapies of GC may be achieved from three different angles, E2F1, E2F7, and MYBL2 themselves, E2F1/E2F7 expression balance, and E2F7 nuclear localization.