Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) has been known to exert tumor suppressive activity for long without much knowledge about its regulation and implications. Protein kinase B (Akt), Protein kinase C (PKC) and Ribosomal protein S6 Kinase (S6K) are known downtargets of PHLPP2, regulating a plethora of life processes viz. cell growth, survival and evasion from apoptosis. Present study decoded the crucial role of PHLPP2 in inducing apoptosis by its interaction with the newly found binding partner Mammalian sterile 20-like kinase 1 (Mst1) in berberine (BBR)-treated human hepatoma cells. HepG2 cells were exposed to (50 μM, 100 μM) berberine for different time intervals (18 h, 24 h). The results showed enhanced expression of PHLPP2 at transcriptional (2.13 fold, P < 0.01) and translational level (4 fold, P < 0.001), but not of PHLPP1, in berberine-treated HepG2 cells. Elevated expression of PHLPP2 was reported to inactivate Akt by dephosphorylating it on Ser473 (P < 0.001). As Akt is known to inhibit apoptotic effect of Mst1, we found that PHLPP2 mediated inactivation of Akt releases its repression from Mst1 leading to heightened phosphorylation of Mst1 on its activating site Thr183 (1.5 fold, P < 0.001). Consequently, coordination between PHLPP2, Akt and Mst1 stimulated downstream targets c-jun N-terminal kinase (JNK), Bim and Bak which are direct activators of pro-apoptotic proteins leading to cell death. Further, PHLPP2/Mst1 knock-down efficiently curtailed anti-proliferative effect of berberine by restoring the basal level of downstream anti-apoptotic proteins. In addition, pre-treatment of NAC (5 mM) showed that ROS generation was a primitive event to initiate activation of stress kinases. Thus, our findings suggest that PHLPP2, Akt and Mst1 constitute an autoinhibitory triangle which may be partly responsible for antiproliferative effect of berberine.