The aims of the study were to gain a better understanding of fructan metabolism regulation during regrowth of Lolium perenne, and to evaluate the role of fructans of remaining tissues as well as carbon assimilation of new leaf tissues in refoliation. Two varieties that contrast for carbohydrate metabolism, Aurora and Perma, were subject to severe and frequent or infrequent defoliations before regrowth. Aurora, which had a greater content of fructans in leaf sheaths than Perma before defoliation, produced more leaf biomass within the 4 days following the first cut. At the end of the regrowth period, Aurora produced more leaf biomass than Perma. Photosynthetic parameters, which were barely affected by defoliation frequency, could not explain these differences. Fructan synthesising activities [sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 6G-fructosyltransferase (6G-FFT)], declined after defoliation. In elongating leaf bases, corresponding transcript levels did not decline concomitantly, suggesting a post-transcriptional regulation of expression, while in leaf sheaths the gene expression pattern mostly followed the time-course of the enzyme activities. Regulation of Lp1-SST and Lp6G-FFT gene expression depends, therefore, on the sink–source status of the tissue after defoliation. During the phase of reserve accumulation, fructosyltransferase activities together with corresponding transcripts increased more in frequently defoliated plants than in infrequently defoliated plants.