Fasciclin-like arabinogalactan proteins (FLAs) have been shown to improve plant tolerance to salt stress. However, their role in cold tolerance (CT) remains unclear. Here, we report that banana MaFLA27 positively regulates CT in Arabidopsis. MaFLA27-overexpression (OE) caused the upregulation of differentially expressed arabinogalactan proteins (AGPs) and genes involved in the biosynthesis of cellulose, lignin, and xylan, as well as the degradation of pectin and xyloglucan. Correspondingly, MaFLA27-OE plants exhibited increased cell wall thickness, enhanced cellulose lignin and starch granule content, elevated levels of partially homogalacturonans recognized by JIM5 and JIM7 antibodies, xyloglucan components recognized by CCRC-M39/104 and LM15 antibodies, LM14 antibody binding AGPs. In contrast, transgenic plants showed a decreased degree of pectin methyl-esterification and accumulated less reactive oxygen species after cold acclimation when compared to wild-type plants. A higher number of pectin methylesterases and cellulose and xylan biosynthesis genes were elevated after cold acclimation. Additionally, both Arabidopsis mutant cesa8 and cellulose inhibitor-treated plants displayed decreased freezing tolerance. Our data suggested that MaFLA27-OE in Arabidopsis may perceive and transmit low-temperature stress signals to the cellulose synthase complexes, activating cellulose synthesis and enhancing cold tolerance. These findings reveal a previously unreported cold-tolerance function of FLAs and highlight associated cell wall-mediated tolerance mechanisms.