Aluminum (Al) toxicity is one of the factors limiting alfalfa production on acid soils. This study aims to investigate the different mechanisms in growth, Al content, pectin content, pectin methylesterase (PME) and the degree of pectin methyl-esterification (DM) in the two root segments of different ecotypes alfalfa during the time of Al stress. It was found interesting that lower root elongation and higher Al content were observed in the Al-sensitive ecotype compared to the Al-resistant ecotype of alfalfa. The Al accumulation increased with the increased of stress time. Meanwhile, the pectin and PME activity of cell wall significantly increased. While DM in Al-sensitive ecotype decreased, more free pectin acid residues on the cell wall of Al-sensitive ecotype. The Al content was higher in 0-5 mm root segments and the cell wall for both materials. The increase in pectin content and PME activity along with the decrease in DM in this segment could determine the alfalfa sensitivity to Al more effectively. The correlation analysis showed that there was a relationship between Al content, pectin content, PME activity of cell wall and DM. The different temporal dynamics and spatial differences caused the increase of Al content, pectin content and cell wall PME activity, changes of DM under Al toxicity cooperates to determine the difference of Al resistance that confers different ecotypes of alfalfa.