Whether lysine dihydroxyisobutyrylation (Khib), a recently identified post-translational modification, is involved in plant responses to low temperature is unknown. Here, we performed a comprehensive chloroplast proteome-wide dihydroxyisobutyrylation analysis in Dendrobium huoshanense to explore the potential function of Khib-modified proteins in response to low-temperature stress. We identified a total of 13,040 Khib sites in 3,281 chloroplast proteins that were significantly differentially regulated by low-temperature stress. Khib-modified proteins were found to be highly associated with proton transmembrane transporter activity, ATPase-coupled ion transmembrane transporter activity, and active monoatomic ion transmembrane transporter activity, among other processes. The identified Khib sites that were found to be differentially regulated in response to low- temperature stress were mainly concentrated in AKhib, KKhib, and EKhib motifs. Notably, D. huoshanense fructose-1,6-bisphosphate aldolase (FBA), an important chloroplast metabolic enzyme involved in the Calvin-Benson cycle, showed significant Khib modification after low-temperature treatment, with eight Khib sites undergoing modification. Site-directed mutagenesis of the DhFBA-encoding gene and its subsequent expression in yeast revealed that Khib modification at the K338 site is essential for maintaining DhFBA enzymatic activity and conferring low-temperature tolerance. Molecular dynamics simulation and surface electrostatic potential analysis further showed that Khib at K338 improved the structural stability of DhFBA by reducing its surface affinity to an optimal status, thus promoting its activity and low-temperature resistance.