Cold stress impedes the growth and development of wheat (Triticum aestivum) and other crops, ultimately reducing both yields and quality. Research indicates that non-coding RNAs (ncRNAs) play a crucial role in regulating plant stress responses and resistance. In a previous study, we observed that the expression of tae-miR164 was inversely correlated with the expression of TaNAC6A in Dongnongdongmai 1 (Dn1), a winter wheat variety with high cold resistance, under cold-stress conditions. However, the molecular mechanism governing the cold responsiveness of the tae-miR164-TaNAC6A module was not fully understood. We found that tae-miR164 and TaNAC6A were both induced to express in opposite trends, and TaNAC6A was located in the nucleus. We also discovered that the expression of tae-miR164 and its target gene, TaNAC6A, was responsive to short-term freezing stress in transgenic Arabidopsis plants. Compared to wild-type (WT) Arabidopsis plants, OE-tae-miR164 plants showed decreased cold tolerance, whereas those overexpressing TaNAC6A demonstrated increased tolerance. On average, the OE-TaNAC6A and STTM-tea-miR164 plants exhibited fewer morphological abnormalities in response to cold stress, higher antioxidant enzyme activities and gene expression levels, lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and higher expressions of AtDREB1, AtDREB2, and AtABI5 in the cold-signaling pathway. Thus, the biological functions of tae-miR164 and TaNAC6A were initially confirmed through heterologous expression strategies, and we have made the first attempt to elucidate its associated tae-miR164-TaNAC6A module of cold resistance. The findings of this research will support further investigations into the regulation of plant stress resistance by ncRNAs and will inform molecular module breeding strategies aimed at enhancing the cold tolerance of crop plants. Molecular module design breeding, as a significant breakthrough in modern biotechnology, is transforming traditional breeding models. Conventional hybrid breeding relies on empirical screening, which is time-consuming and subject to randomness. In contrast, molecular module breeding directly targets key genes and achieves precise regulation through technologies such as gene editing and synthetic biology.