Plants are often exposed to fluctuating light under natural conditions, but the effects of changing frequency of fluctuating light on photosynthesis and related mechanism are not clear. In order to study the photosynthetic mechanism under fluctuating light, the changes of morphology, chlorophyll content, gas exchange, chlorophyll a fluorescence and enzymatic antioxidant system were compared in cucumber seedlings grown under different light regimes. Results showed that the plant height, biomass, leaf area and specific leaf weight were significantly higher under high light treatment (T1, T2 and T3) than those of low light treatment (T4), and these values decreased with the frequency of fluctuating light increasing (T2 and T3). Compared with low light treatment, the total chlorophyll content decreased under high light, and enhancing the frequency of fluctuating light resulted in the decrease in the chlorophyll content slightly. Photosynthetic rate and stomatal conductance in leaves grown under high light were higher than those under low light, and the levels of both parameters decreased slightly with the changing frequency of fluctuating light increasing. The results of chlorophyll a fluorescence induction kinetics showed that although there was no significant difference in the maximum quantum yield of photosystem II (PSII), the electron transport activity of PSII in cucumber leaves decreased with the increase of the changing frequency of fluctuating light. Increasing the changing frequency of fluctuating light led to an obvious decrease in antenna conversion efficiency of PSII ( F v′/ F m′) and rapid increase in non-photochemical quenching (NPQ). In addition, the activities of the major antioxidant enzymes in cucumber leaves grown under high light was higher than that under low light, yet the increase of the frequency of fluctuating light reduced their activities. Therefore, enhancing the changing frequency of fluctuating light leads to a decrease in photosynthetic rate and the activities of major antioxidant enzymes in cucumber seedlings, the enhanced thermal dissipation may play a very important role in copying with excessive excited energy under fluctuating light. In addition, the difference in acclimative mechanism between fluctuating and steady-state high light in cucumber seedlings were also discussed.