Although the regulation in Pi homeostasis of miR399 have been studied in various plants, its molecular mechanisms in response to freezing stress are still elusive. In this work, we found that the expression of tae-miR399 and its target gene TaUBC24 in tillering nodes of strong cold resistance winter wheat cultivar Dongnongdongmai1 (Dn1) was significantly altered subjected to severe winter. tae-miR399 and its target gene TaUBC24 were also responsive to short-term freezing stress in tillering nodes of Dn1 seedlings. TaUBC24 physically interacted with TaICE1. Ehanced freezing tolerance was observed in overexpressing tae-miR399 Arabidopsis lines. Under freezing stress, overexpressing tae-miR399 decreased the expression of AtUBC24 to increase the expression of genes in CBF signaling pathway, Pi translocation pathway and starch metablism, including AtCBFs, AtCOR47, AtCOR413IM, AtPHT1;4, AtAPLs and AtBAMs, inhibit the degradation of AtICE1 and AtPHO1, and promote the activities of SOD, POD and CAT. These findings indicated that the increased freezing tolerance was dependent upon elevating CBF signaling pathway, phosphorus utilization efficiency, starch degradation, accumulation of soluble sugar and ability of ROS scavenge. These results will aid our understanding of molecular mechanism of how miR399-UBC24 module plays a cardinal role in regulating plant freezing stress tolerance through mediating the downstream pathways.