Very little is known about the mechanism controlling petiole rigidity in sacred lotus (Nelumbo nucifera Gaertn.). To investigate the mechanism controlling the lotus petiole rigidity, morphological and proteomic analyses were performed. Anatomically, there is a great variation between the petioles of floating and vertical leaves. The number of vascular bundles, ligneous cells and thickness of cell wall were higher in the initial vertical leaf petiole (IVP) compared to the initial floating leaf petiole (IFP). A total of 4855 proteins were quantified through comparative proteomic analysis, among which 421 proteins expressed 1.5 folds higher in IFP and 483 proteins expressed 1.5 folds higher in IVP. Protein function categories indicated hundreds of proteins involved in cell wall biosynthesis, organization and assembly. Functional enrichment analysis for the differentially abundant proteins indicated the enrichment of 105 proteins in 6 different pathways, while 43 out of them were enriched in lignin biosynthesis pathway. In consistent with genes and proteins expressions in lignin biosynthesis, the contents of lignin monomers precursors were significantly different in IFP and IVP. These findings support the involvement of lignin in lotus petioles rigidity.