The temperature drop of plants from the optimal requirements can increase tolerance to severe chilling stress. Photosynthesis and osmoregulators were analyzed during chilling stress to explore the adaptation mechanisms that underlie the induction of chilling tolerance in response to suboptimal temperature. The relationships of these processes to suboptimal temperature acclimation in watermelon were then determined. Suboptimal temperature-acclimated watermelon plants demonstrated tolerance during chilling stress, as indicated by the decreased electrolyte leakage and malondialdehyde accumulation compared with those non-acclimated watermelon plants. Chilling-induced photoinhibition and reduction in CO2 assimilation rate were alleviated after suboptimal temperature acclimation. The xanthophyll cycle level was enhanced by improving thermal dissipation ability and avoiding light damage. Consequently, the chilling tolerance of suboptimal temperature-acclimated watermelon plants was enhanced. The osmoregulation ability induced by suboptimal temperature acclimation protected watermelon plants against chilling injury because of the accumulation of small molecular substances, such as soluble sugar and proline. The protein levels of Rubisco activase (ClRCA) and the gene expression of the Benson–Calvin cycle simultaneously increased in suboptimal temperature-acclimated watermelon plants during chilling stress. Chilling tolerance in watermelon plants induced by suboptimal temperature acclimation is associated with enhanced photosynthetic adaptability and osmoregulation ability. Keywords: Citrullus lanatus, Chilling stress, Acclimation, Photosynthesis, Osmoregulation, ClRCA