To obtain new insights into the mechanisms through which melatonin reduces photoinhibition in cucumber seedlings during chilling, we investigated its effects on the Calvin-Benson cycle. Photoinhibition due to chilling was significantly reduced by prior irrigation with a 200 μM melatonin solution. This was evidenced by reduced declines in the quantum yield of photochemical energy conversion in PS II [Φ(PSⅡ)], in the rate of electron transport through PSII [Je(PSII)] and in the electron flux in the PCR cycle [Je(PCR)], and an increase in the PSII excitation pressure (1-qP). The reasons for this can be explained as: (1) melatonin upregulates the expressions of CsrbcL and CsrbcS, and lessens the decrease in Rubisco content induced by chilling; (2) melatonin upregulates the expression of CsRCA, increases the content of Rubisco activase (RCA), and promotes the transformation of Rubisco from the inactive state to the active state, thereby increasing the carboxylation efficiency of Rubp and helping maintain normal rates of CO2 fixation; and (3) melatonin upregulates the expressions of key genes in the Rubp regeneration process to help maintain the regeneration of Rubp required for CO2 fixation, and thus the normal operation of the Calvin-Benson cycle. This study provides new evidence that suggests melatonin reduces photoinhibition during chilling by regulating the Calvin-Benson cycle.