Protamines are a group of highly basic proteins first discovered in spermatozoon that allow for denser packaging of DNA than histones and will result in down-regulation of gene transcription[1]. It is well recognized that the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes P6.9, a protamine-like protein that forms the viral subnucleosome through binding to the viral genome[29]. Previous research demonstrates that P6.9 is essential for viral nucleocapsid assembly, while it has no influence on viral genome replication[31]. In the present study, the role of P6.9 in viral gene transcription regulation is characterized. In contrast to protamines or other protamine-like proteins that usually down-regulate gene transcription, P6.9 appears to up-regulate viral gene transcription at 12-24 hours post infection (hpi), whereas it is non-essential for the basal level of viral gene transcription. Fluorescence microscopy reveals the P6.9's co-localization with DNA is temporally and spatially synchronized with P6.9's impact on viral gene transcription, indicating the P6.9-DNA association contributes to transcription regulation. Chromatin fractionation assay further reveals an unexpected co-existence of P6.9 and host RNA polymerase II in the same transcriptionally active chromatin fraction at 24 hpi, which may probably contribute to viral gene transcription up-regulation in the late infection phase.