In sugarcane, increased sink demand has previously been shown to result in increased photosynthetic rates that are correlated with a reduction in leaf hexose concentrations. To establish whether sink limitation of photosynthesis is a result of sugar accumulation in the leaf, excision and cold-girdling techniques were used to modify leaf sugar concentrations in pot-grown sugarcane. In excised leaves that were preincubated in darkness for 3 h, sucrose accumulation was reduced but accumulated again upon transfer to the light, while hexose concentrations remained lower than in controls (7.7 μmol mg−1 FW versus 18.6 μmol mg−1 FW hexose in controls). These results were associated with a 66% and 59% increase in photosynthetic assimilation (A) and electron transport rate (ETR), respectively, compared to controls maintained in the light. Similar increases in photosynthesis were observed when dark-treated leaves were supplied with 5 mM sorbitol, but not when supplied with 5 mM sucrose. Further analyses of 14C-labeled sugars indicated rapid turnover between sucrose and hexose. Cold-girdling (5 °C) increased sucrose and hexose levels and resulted in a decline of photosynthetic rates over 5 d (48% and 35% decline in assimilation rate and ETR, respectively). These sugar-induced changes in photosynthesis were independent of changes in stomatal conductance. This study demonstrates that the down-regulation of photosynthesis in response to culm sugar accumulation reported previously could be due to the knock-on effect of accumulation of sugar in leaf tissue, and supports the contention that hexose, rather than sucrose, is responsible for the modulation of photosynthetic activity.