Lysine crotonylation of histone proteins is a newly identified post-translational modification with diversified cellular functions. However, there’s few reports on lysine crotonylation of non-histone proteins in fruit plant cells. By using high-resolution LC-MS coupled with highly sensitive specific immune-affinity antibody analysis, a whole crotonylation proteome analysis of Dendrobium huoshanese was performed. In total, 1,591 proteins with 4,725 lysine crotonylation sites were found, among them eleven conserved motifs have been identified. Bioinformatic analysis linked crotonylated proteins to multiple metabolic pathways, including secondary metabolites biosynthesis, transport and catabolism; energy production and conversion; carbohydrate transport and metabolism; and translation, ribosomal structure and biogenesis.