Summary. Many insect species overcome unfavourable environmental conditions by entering diapause, a type of hypometabolic state of arrested development during which they become vulnerable and sensitive to microbial infections, especially in light of ongoing climate changes. In order to estimate how prolonged heat stress during the winter diapause of Ostrinia nubilalis (Hübner) affects the insect’s immune system, diapausing larvae were reared from November 2018 until May 2019 in corn stalks under different thermal conditions – field, with ambient winter temperatures and laboratory, with above-average temperatures (12-18 °C). Changes in O. nubilalis immune response have been assessed at the transcriptional level, by analyzing the expression of genes encoding two major antimicrobial peptides, attacin and gloverin, using qPCR. Total RNA was isolated from whole-body homogenates of diapausing 5th instar larvae reared at different temperatures and non-diapausing larvae and pupae, as control groups. Relative gene expression was determined using actin as the reference gene. During early-diapause, the relative expression of attacin and gloverin was suppressed in diapausing larvae under both thermal regimes, in comparison to non-diapausing larvae and pupae. However, downregulation of gene transcription was prolonged and more profound in heat stressed diapausing larvae, in comparison to larvae from field conditions. Also, larvae resting in field conditions had higher transcriptional activity of both analyzed antimicrobial genes, especially from mid-diapause and onwards. Heat stressed larvae only exhibited a significant peak in gene expression during March, probably as a consequence of their increased immune response due to pathophysiological processes that led to high larval mortality and premature diapause termination in early April. Moreover, recorded transcriptional suppression in the early months of the resting state corresponds to general metabolic depression, probably as part of the diapausing program. The gradual increase in relative expression of both antimicrobial peptides during the latter months of diapause in larvae from field conditions can be correlated with the termination of their resting period and preparation of larval immune system for active development and subsequent metamorphosis.