Spinach (Spinacia oleracea) plants were grown under the day/night temperature regime of 15/10 °C (LT) or 30/25 °C (HT). The plants were also transferred from HT to LT when the sample leaves were at particular developmental stages (HL-transfer). With fully mature leaves, the light-saturated photosynthetic rate (A) at the ambient CO2 concentration (Ca) of 1500 µL L−1 (A1500) and the initial slope of A versus intercellular CO2 concentration (Ci) at low Ci region (IS) were obtained to assess capacities of RuBP regeneration and carboxylation. Photosynthetic components including Rubisco and cytochrome f (Cyt f) were also determined. The optimum temperatures for A at Ca of 360 µL L−1 (A360), A1500 and IS in HT leaves were 27, 36 and 24 °C, whereas those in LT leaves were 18, 30 and 18 °C. The optimum temperatures in HL-transfer leaves approached those of LT leaves with the increase in the duration at LT. The shift in the optimum temperature was greater and quicker for IS than A1500. By the HL-transfer, the maximum values of A1500 and IS also increased. The maximum A1500 and Cyt f content increased more promptly than IS and Rubisco content. Changes in the Cyt f/Rubisco ratio were reflected to those in the A1500/IS ratio. Taken together, photosynthetic acclimation to low temperature in spinach leaves was due not only to the change in the balance of the absolute rates of RuBP regeneration and carboxylation but also to the large change in the optimum temperature of RuBP carboxylation.