Background Rhizome is the storage underground stem of lotus (Nelumbo nucifera), which is enlarged before winter season and could be used for asexual propagation. In addition, the enlarged rhizome is a nutritional vegetable with abundant starch, proteins, and vitamins. Enlargement of lotus rhizome is not only significance for itself to survive from the cold winter, but also important for its economic value. Results To explore the mechanism underlying its enlargement, integrative analyses of morphology, physiology and proteomics were conducted on the rhizome at stolon, middle, and enlarged stages. Morphological observation and physiological analyses showed that rhizomes were gradually enlarged during this process, in which the starch accumulation was also initiated. Quantitative proteomic analysis on the rhizomes at these three stages identified 302 stage-specific proteins (SSPs) and 172 differently expressed proteins (DEPs), based on which GO and KEGG enrichment analyses were conducted. The results indicated that light and auxin signal might be transduced through secondary messenger Ca2+, and play important roles in lotus rhizome enlargement. Conclusion These results will provide new insights into understanding the mechanism of lotus rhizome enlargement. Meanwhile, some candidate genes might be useful for further studies on this process, as well as breeding of rhizome lotus.