Recently, we have demonstrated that microRNA-31 (miR-31) overexpression is inherent to radiation-induced cell death in the highly radioresistant Sf9 insect cells, and regulates pro-apoptotic Bax translocation to mitochondria. In the present study, we report that at sub-lethal radiation doses for Sf9 cells, miR-31 is significantly downregulated and is tightly regulated by an unusual mechanism involving p53. While ectopic overexpression of a well-conserved Sfp53 caused typical apoptosis, radiation-induced p53 accumulation observed selectively at sub-lethal doses failed to induce cell death. Further investigation of this paradoxical response revealed an intriguing phenomenon that sub-lethal radiation doses result in accumulation of a 'hyper-phosphorylated' Sfp53, which in turn binds to miR-31 genomic location and suppresses its expression to prevent cell death. Interestingly, priming cells with sub-lethal doses even prevented the apoptosis induced by lethal radiation or ectopic Sfp53 overexpression. On the other hand, silencing p53 increased radiation-induced cell death by inhibiting miR-31 downregulation. This study thus shows the existence of a unique radiation-responsive 'p53 gateway' preventing miR-31-mediated apoptosis in Sf9 cells. Since Sfp53 has a good functional homology with human p53, this study may have significant implications for effectively modulating the mammalian cell radioresistance.