The root barrier to radial O2 loss (ROL) is a key root trait preventing O2 loss from roots to anoxic soils, thereby enabling root growth into anoxic, flooded soils. We hypothesized that the ROL barrier can also prevent intrusion of hydrogen sulphide (H2S), a potent phytotoxin in flooded soils. Using H2S- and O2-sensitive microsensors, we measured the apparent permeance to H2S of rice roots, tested whether restricted H2S intrusion reduced its adverse effects on root respiration, and whether H2S could induce the formation of a ROL barrier. The ROL barrier reduced apparent permeance to H2S by almost 99%, greatly restricting H2S intrusion. The ROL barrier acted as a shield towards H2S; O2 consumption in roots with a ROL barrier remained unaffected at high H2S concentration (500 μM), compared to a 67% decline in roots without a barrier. Importantly, low H2S concentrations induced the formation of a ROL barrier. In conclusion, the ROL barrier plays a key role in protecting against H2S intrusion, and H2S can act as an environmental signalling molecule for the induction of the barrier. This study demonstrates the multiple functions of the suberized/lignified outer part of the rice root beyond that of restricting ROL.