Abstract: Phytochrome-interacting factors (PIFs), a subfamily of basic helix-loop-helix (bHLH) transcription factors, are key regulators of plant development and environmental adaptation. Low temperatures represent a major constraint on grapevine cultivation in temperate regions. In this study, VaPIF3b was cloned from Vitis amurensis Rupr. cDNA based on transcriptomic data. Bioinformatics and transient silencing of grapevine plants were performed to explore the cold resistance function and mechanism of VaPIF3b. Results showed that the VaPIF3b transcript is 1 725 bp in length and encodes 574 amino acids, with a predicted molecular weight of 61.90 kDa, an isoelectric point (pI) of 8.44, an average hydrophilicity of −0.487, and a protein instability coefficient of 52.07, classifying it as an unstable hydrophilic protein. Subcellular localization assays confirmed the nuclear targeting of VaPIF3b. Yeast transcriptional assays revealed that VaPIF3b lacks intrinsic transcriptional activation activity. Silencing VaPIF3b using VIGS technology reduced grapevine cold tolerance. Dual-luciferase analysis demonstrated that VaPIF3b can bind to the VaCBF4 promoter and activate its expression. These findings indicate that VaPIF3b modulates grapevine cold stress response via activation of the CBF-dependent signaling pathway.