The photosynthetic rate may be strongly limited by internal conductance from the intercellular airspace to the chloroplast stroma (gi). However, the effects of growth and leaf temperature on gi are still unclarified. In this work, we determined the temperature dependence of gi in spinach leaves grown at 30/25°C (high temperature; HT) and 15/10°C (low temperature; LT), using the concurrent measurements of the gas exchange rate and stable carbon isotope ratio. Moreover, we quantified the effects of gi on the temperature dependence of the photosynthetic rate. We measured gi and the photosynthetic rate at a CO2 concentration of 360 μl l−1 under saturating light (A360) at different leaf temperatures. The optimum temperature for A360 was 28.5°C in HT leaves and 22.9°C in LT leaves. The optimum temperatures for gi were almost similar to those of A360 in both HT and LT leaves. There was a strong linear relationship between A360 and gi. The photosynthetic rates predicted from the C3 photosynthesis model taking account of gi agreed well with A360 in both HT and LT leaves. The temperature coefficients (Q10) of gi between 10 and 20°C were 2.0 and 1.8 in HT and LT leaves, respectively. This suggests that gi was determined not only by physical diffusion but by processes facilitated by protein(s). The limitation of the photosynthetic rate imposed by gi increased with leaf temperature and was greater than the limitation of the stomatal conductance at any temperature, in both HT and LT leaves. This study suggests that gi substantially limits the photosynthetic rate, especially at higher temperatures.