BACKGROUND: Alfalfa (Medicago sativa L.) is a high-quality, high-protein forage, and the improvement and breeding of key traits are important for enhancing the productivity of alfalfa. Plant height is an important trait that affects crop yield, and its regulatory network mechanism has been widely reported in model plants, however, there are fewer studies on the developmental regulatory of plant height in alfalfa. RESULTS: In this study, we screened tall (WL525HQ) and short (WL343HQ) alfalfa materials through field experiments and analyzed the regulatory mechanism of plant height based on the multidimensional joint analysis of phenotype, cell, physiology, and molecular biology. The results showed that internode length was an important factor determining plant height in alfalfa, and cell size affected the internode elongation to a certain extent, whereas cell size was limited by cell wall. Moreover, changes in cell wall components play an important role in cell wall expansion, especially lignin synthesis. Transcriptome analysis showed that the high expression of hydrolase activity in T1 (initiation growth period) facilitates the expansion of the cell wall, the significant enrichment of the cellular modification process in T3 (rapid growth period) increases the cell size, and the synthesis of cell wall structural constituents and plant-type cell wall organization in T5 (growth stabilization) further improves and modifies the cell wall structure. Differential genes involved in cell wall biosynthesis and expansion were mainly enriched in cellulose synthesis, pectin cleavage, lignin formation, expansion protein (EXP), and xyloglucan endotransglycosidase (XTH). CONCLUSIONS: These findings elucidated the plant height regulation mechanisms throughout the alfalfa plant and provided a theoretical basis for the generation of ideal alfalfa plant height germplasm.