The polar fungus Geomyces sp. WNF-15A produces high-quality red pigment (AGRP), but the cold-dependent characteristic of AGRP synthesis restricts its industrialization. This study employed transcriptome analysis to compare gene expression profiles of the wild-type strain with cold-independent mutants of scaffold1.t692 (Δ1-692) and scaffold2.t704 (Δ2-704). From the analysis, 23 candidate genes were identified and functionally characterized among 22,600 differentially expressed genes. Knockout and recovery of scaffold5.t61, scaffold7.t586, or scaffold7.t712 proved their regulatory functions in AGRP synthesis, among which scaffold5.t61 functioned as a transcription factor, while scaffold7.t586 and scaffold7.t712 were involved in the glutamate-related nitrogen metabolism. Exogenous addition of nitrate, glutamine, and glutamate, combined with transcriptional regulation studies, revealed the importance of glutamate metabolism for cold-adaptive synthesis of AGRP. Scaffold5.t61 responded to the cold environment and regulated the transcription of scaffold2.t704 and scaffold1.t692. It subsequently increased glutamate synthesis by regulating the key nitrogen metabolism genes of scaffold7.t586 and scaffold7.t712, ultimately resulting in cold-dependent synthesis of AGRP in Geomyces sp. WNF-15A. This study offers new insights into the mechanisms of cold adaptation in polar fungi and serves as a reference for the development of psychrophilic fungal resources.