We previously identified a large homologous region (hr), hycu-hr6, in the genome of the Hyphantria cunea nucleopolyhedrovirus (HycuNPV) and suggested that hycu-hr6 was the largest baculovirus promoter enhancer hr identified so far. In this study, we examined the enhancement activity of hycu-hr6 against two promoters from constitutive baculovirus immediate early genes, the HycuNPV ie1 (hycu-ie1) and the Orgyia pseudotsugata multicapsid (M) NPV ie2 (op-ie2), and against a promoter from the inducible Drosophila heat shock protein 70 gene (hsp70) in five lepidopteran (BmN-4, Ld652Y, Sf9, SpIm, and TN368) and one dipteran (S2) insect cell lines. Comparative analyses of transient expression assays using the firefly luciferase gene (luc) as a reporter showed that hycu-hr6 enhanced the activity of all three promoters in all tested cell lines. Comparison of the enhancement efficiency of hycu-hr6 with that of Autographa californica MNPV (AcMNPV) hr5, an enhancer commonly used to improve the activity of AcMNPV ie1 promoter in many insect expression vectors, established that hycu-hr6 was a more efficient enhancer. These results indicate that hycu-hr6 is a versatile and superior promoter enhancer in several insect cell lines and favor the incorporation of hycu-hr6 into insect expression vectors to maximize promoter activity.