Abstract The present study explored the role of calcium ion (Ca2+) in nitric oxide (NO)-induced tolerance to low temperature in cucumber (Cucumis sativus L.) seedlings. Low temperature (11 °C/7 °C) induced a raise in NO accumulation and caused significant damages to photosynthetic processes in cucumber leaves, as evidenced by the decreased net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), chlorophyll content, maximal photochemical efficiency of photosystem II (PSII) (Fv/Fm), maximal fluorescence (Fm) photochemical quantum yield [Y (II)], relative apparent electron transport rate (ETR), quantum yield of PSII electron transport (Fm/Fo) and latent PSII quantum yield (Fv/F0), and the increased intercellular CO2 concentration (Ci), parameters of quantum yield of regulated energy dissipation [Y (NPQ)], and quantum yield of non-regulated energy dissipation [Y (NO)]. However, exogenous sodium nitroprusside (SNP), a donor of NO, ameliorated the negative effects of low temperature. Furthermore, the content of starch, sucrose, glucose, fructose, soluble sugar and reducing sugar, as well as the transcript levels of subunit of magnesium chelatase (ChlD, ChlI, ChlH), chlorophyll a-b binding protein (Chl), original chlorophyll redox enzymes (POR) and cytochrome b6/f complex (Cytb6f) genes were elevated by the treatment with SNP alone, whereas the inhibition of Ca2+ with EGTA (Ca2+ chelating agent), LaCl3 (Ca2+ channel blocker), TFP and W-7 (calmodulin antagonists) attenuated or almost abolished the aforementioned effects of SNP under low temperature. Taken together, our findings demonstrated that Ca2+ participated in the NO-induced tolerance to low temperature by modulating the leaf gas exchange, processes of PSII, carbohydrate metabolism and expression of chlorophyll synthesis-related genes in cucumber leaves.