Defoliation often has little effect on the percent plant root length colonised by arbuscular mycorrhizal (AM) fungi, and this has been interpreted as a lack of support for the carbon limitation hypothesis. We performed an experiment with three levels of repeated defoliation (none, every 3 weeks and weekly) of Lolium rigidum growing in pasture soil, and assessed colonisation of roots by naturally occurring AM fungi over 4 months in the glasshouse. Surprisingly, the percent root length colonised by AM fungi increased with defoliation. We also assessed root mass and the length of colonised root to obtain an estimate of the quantity of mycorrhizal root. As expected, both root mass and length of mycorrhizal root decreased with defoliation, as did soluble sugars in the roots. Thus, increasing the frequency of defoliation reduced mycorrhizas consistent with the carbon-limitation hypothesis. Such a decline in mass of mycorrhizal root could contribute to reducing soil biological fertility in pastures over time.