Leafy vegetables cultivated in greenhouses during the winter are sometimes exposed to cold air from outside the greenhouse to enhance sugar and nutrient content. To analyze the possible involvement of photosynthetic and respiratory activities in this process, we evaluated the gas-exchange activity of spinach (Spinacia oleracea L.) plants cultivated in an unheated greenhouse in mid-winter in Sapporo, where the daily mean air and soil temperatures are approximately -5 and 0 °C, respectively. Shoot fresh weight showed little increase, whereas the net leaf photosynthetic rate (Pn) attained 20 µmol m-2 s-1 and the CO2 concentration in the greenhouse ([CO2]) was sometimes lower than 200 µmol mol-1, which was suggestive of active photosynthetic CO2 uptake. After its peak in the morning, Pn decreased in the afternoon, presumably owing to ‘midday depression’ caused by suppressed water uptake in the root zone. Observed diurnal [CO2] change was consistent with a significant CO2 uptake during the daytime. The change also suggested that respiration was active immediately after sunset and suppressed at night. In addition, we calculated the whole-greenhouse CO2 emission rate (R) as a measure of night respiration in the plants, taking into account the air ventilation of the greenhouse. The R value was positive under sub-zero air temperatures in the greenhouse and was positively correlated with the nighttime air and soil temperatures. These experimental data suggest active photosynthesis and respiration of winter-sweetened spinach in the greenhouse, despite the low air and soil temperatures and growth retardation, and implies their involvement in the sweetening process.