Fruit antioxidants are necessary for human health and also have physiological roles in protecting fruit against oxidative stress. Our previous work has shown that monodehydroascorbate reductase (MDHAR) activity was positively correlated with fruit resistance to cold storage in introgression lines of tomato, Solanum lycopersicum. We have produced transgenic lines with reduced MDHAR activity to directly study the impact of this gene on fruit resistance to chilling and the antioxidant pool in two different genetic backgrounds: M82, a processing tomato with low to medium ascorbate levels, and IL925, an introgression line of M82 containing a fragment of the wild tomato Solanum pennellii genome, including the S. pennelli MDHAR allele. Large decreases in fruit MDHAR activity were obtained and correlated with slight losses in fruit firmness and fruit ascorbate, an effect that was independent of the genetic background. Fruit colour after chilling was also positively correlated with the redox state of the ascorbate pool and fruit firmness, although at harvest these correlations were less significant. Furthermore, other modifications in fruit of transgenic lines were observed as red fruit showed increased glutathione levels. Therefore manipulation of MDHAR activity confirms the link between antioxidant protection, fruit tolerance to chilling and fruit ripening at low temperatures, although the strength of the phenotypes indicates that other factors are involved.