© 2016 Elsevier Ireland Ltd.Lotus species are important forage legumes due to their high nutritional value and adaptability to marginal conditions. However, the dry matter production and regrowth rate of cultivable Lotus spp. is drastically reduced during colder seasons. In this work, we evaluated the chilling response of Lotus japonicus ecotypes MG-1 and MG-20. No significant increases were observed in reactive oxygen species and nitric oxide production or in lipid peroxidation, although a chilling-induced redox imbalance was suggested through NADPH/NADP+ ratio alterations. Antioxidant enzyme catalase, ascorbate peroxidase, and superoxide dismutase activities were also measured. Superoxide dismutase, in particular the chloroplastic isoform, showed different activity for different ecotypes and treatments. Stress-induced photoinhibition also differentially influenced both ecotypes, with MG-1 more affected than MG-20. Data showed that the D2 PSII subunit was more affected than D1 after 1 d of low temperature exposure, although its protein levels recovered over the course of the experiment. Interestingly, D2 recovery was accompanied by improvements in photosynthetic parameters (Asat and Fv/Fm) and the NADPH/NADP+ ratio. Our results suggest that the D2 protein is involved in the acclimation response of L. japonicus to low temperature. This may provide a deeper insight into the chilling tolerance mechanisms of the Lotus genus.