Studies have shown that inhibition of PI3K/AKT signaling is a key strategy for the treatment of tyrosine kinase inhibitor resistance in non-small cell lung cancer (NSCLC). Vasculogenic mimicry (VM) not only accelerates tumor progression but also increases drug-induced resistance. As a tumor suppressor, protein phosphatase 2A (PP2A) is a ubiquitous conserved serine/threonine phosphatase. While its effects and mechanisms on VM formation and invasion in NSCLC remain unclear. The present study aimed to investigate the role of PP2A in VM formation and elucidate the underlying mechanisms. Results showed that PP2A could significantly inhibit VM formation and VM-dependent behavior, including invasion and migration both in vitro and in vivo. Activation of PP2A with FTY720 or Ad-PP2A reduced phosphorylated AKT and inhibited ZEB1 transcription, thereby further downregulating the expression of MMP-2, VE-cadherin, and VEGFR-2, whereas inhibition of PP2A with okadaic acid (OA) or Ad-dn-PP2A exerted the opposite effect. Furthermore, PP2A inhibited tumor growth and VM formation in the xenograft tumor model. PI3K inhibitor BENC-511 could potentiate activation of PP2A, leading to inhibition of p-AKT/ZEB1 and VM formation in vitro and in vivo. This study indicated that PP2A could regulate VM formation in NSCLC through the PI3K/AKT/ZEB1 axis. PP2A reactivation or combination with PI3K inhibitor might be a more effective treatment against advanced NSCLC by inhibiting VM formation.