Magnesium (Mg) supplementation has shown to modulate the stress responses to transport in other species. An experiment was designed to evaluate the effect of Mg for alleviating stress associated with road transportation in lambs. Two groups (n = 18 each) of 4-month old male crossbred lambs (24 ± 4.5 kg) were fed a basal diet comprising oaten hay, pellets and barley, that contained 0.17% Mg. One group was supplemented with magnesium oxide, such that dietary Mg was 0.41%. At the end of the 2-week supplementation period, the lambs were transported by road for 8 h. Blood samples were collected before supplementation, 1 h before transport, within 0.5 h post-transport, at 2 h post-transport and 5 days post-transport. Data were analysed using a repeated-measure analysis of variance and linear mixed model with treatment within time interaction. Supplementation with dietary Mg in the form of MgO increased serum Mg concentrations (1.16 mmol/L ± 0.012, compared with 1.09 mmol/L ± 0.013 in the control group; P < 0.05), but had no effect on alleviating stress during transportation. Transport resulted in a significant increase in serum cortisol, which returned to pre-transport levels 2 h after transport. The serum ÃŸ-hydroxybutyrate was significantly higher 5 days after transport. The post-transport average feed intake and the average daily gain were significantly lower than pre-transport values, suggesting that the stress of transportation resulted in a decrease in feed intake post-transport, which could have implications on liveweight gain of animals after transportation.