Abstract <p>The baculovirus insect cell expression system is pivotal for exogenous protein expression. However, serial passages of baculovirus in insect cells often result in defective virus generation and a rapid decline in exogenous protein expression, limiting its wider application. Previous studies have shown that the expression of the late expression factor 5 (<i>lef5</i>) from other baculoviruses can enhance the stability of exogenous genes in <i>Autographa californica multiple nucleopolyhedrovirus</i> (AcMNPV). In this study, we engineered diverse recombinant strains of AcMNPV to express the enhanced green fluorescent protein (eGFP) and enterovirus 71 virus-like particles (EV71-VLPs), co-expressed with the P1 and 3CD proteins of EV71. We investigated the influence of <i>lef5</i> overexpression, regulated by various promoters, on the stability of exogenous genes and their protein expressions. Notably, <i>lef5</i> overexpression significantly improved the stability of <i>eGFP</i> and <i>EV71-P1</i> gene and protein expressions during serial passages in Sf9 cells. Specifically, the <i>lef5</i> overexpression driven by the op166 promoter was more beneficial for enhancing the expression stability of complex exogenous proteins, such as EV71-VLPs. This study underscores the importance of <i>lef5</i> overexpression in maintaining the stability of exogenous gene expression in baculovirus systems, particularly for producing complex proteins such as VLPs of enterovirus.</p> Key points <p>•&#xa0;<i>The overexpression of lef5 stabilizes exogenous gene expression in the baculovirus system.</i></p> <p>•&#xa0;<i>Promoter choice affects lef5-mediated gene expression stability.</i></p> <p>•&#xa0;<i>The overexpression of lef5 is crucial for producing complex proteins like EV71-VLPs.</i></p>