•Temperature variations significantly influence the growth and metabolite production of C. militaris.•Responsive mechanisms in acclimatization to temperature conditions identified in C. militaris.•Low temperature induced transcriptional upregulation of lipid biosynthetic pathways indicating an adaptive metabolic response of C. militaris. Cordyceps militaris is a medicinal entomopathogenic fungus containing valuable biometabolites for pharmaceutical applications. Its genetic inheritance and environmental factors play a crucial role in the production of biomass enriched with cordycepin. While temperature is a crucial controlled parameter for fungal cultivation, its impacts on growth and metabolite biosynthesis remains poorly characterized. This study aimed to investigate the metabolic responses and cordycepin production of C. militaris strain TBRC6039 under various temperature conditions through transcriptome analysis. Among 9599 expressed genes, 576 genes were significantly differentially expressed at culture temperatures of 15 and 25 °C. The changes in the transcriptional responses induced by these temperatures were found in several metabolisms involved in nutrient assimilation and energy source, including amino acids metabolism (e.g., glycine, serine and threonine metabolism) and lipid metabolism (e.g., biosynthesis of unsaturated fatty acids and steroid biosynthesis). At the lower temperature (15 °C), the biosynthetic pathways of lipids, specifically ergosterol and squalene, were the target for maintaining membrane function by transcriptional upregulation. Our study revealed the responsive mechanisms of C. militaris in acclimatization to temperature conditions that provide an insight on physiological manipulation for the production of metabolites by C. militaris.