MicroRNAs (miRNAs) are non-coding regulatory molecules that play important roles in a variety of biological processes. Although a number of cold-responsive miRNAs have been computationally identified, functions and mechanisms of most miRNAs are not well understood. Herein, the function of trifoliate orange [Poncirus trifoliata (L.) Raf.] miRNA396b (ptr-miR396b) in cold tolerance and its potential regulatory module were investigated. Compared with the wild type (WT), transgenic lemon (Citrus limon) plants overexpressing ptr-MIR396b, the precursor of ptr-miR396b, displayed enhanced cold tolerance. Ptr-miR396b was experimentally confirmed to guide the cleavage of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO). The overexpressing lines exhibited a reduction in ACO transcript levels and ethylene content compared with the WT, and the expression pattern of ACO was opposite to that of ptr-miR396b in response to cold stress. In addition, the transgenic lines exhibited higher levels of free polyamines and mRNA abundance of polyamine biosynthetic genes than WT plants under cold treatment, consistent with reduced reactive oxygen species (ROS) accumulation in the former. Taken together, this study demonstrates that ptr-miR396b positively regulates cold tolerance through reducing ACO transcript levels, thereby repressing ethylene synthesis and simultaneously promoting polyamine synthesis, leading to enhanced ROS scavenging. Identification of the ptr-miR396b–ACO regulatory module provides new insights into the molecular mechanism underlying the reduction of ethylene production under cold.