To test the hypothesis that the up-regulation of sucrose biosynthesis during cold acclimation is essential for the development of freezing tolerance, the acclimation responses of wild-type (WT) Arabidopsis thaliana (Heynh.) were compared with transgenic plants over-expressing sucrose phosphate synthase (over-sps) or with antisense repression of either cytosolic fructose-1,6-bisphosphatase (antifbp) or sucrose phosphate synthase (antisps). Plants were grown at 23 °C and then shifted to 5 °C. The leaves shifted to 5 °C for 10 d and the new leaves that developed at 5 °C were compared with control leaves on plants at 23 °C. Plants over-expressing sucrose phosphate synthase showed improved photosynthesis and increased flux of fixed carbon into sucrose when shifted to 5 °C, whereas both antisense lines showed reduced flux into soluble sugars relative to WT. The improved photosynthetic performance by the over-sps plants shifted to 5 °C was associated with an increase in freezing tolerance relative to WT (−9.1 and −7.2 °C, respectively). In contrast, both antisense lines showed impaired development of freezing tolerance (− 5.2 and −5.8 °C for antifbp and antisps, respectively) when shifted to 5 °C. In the new leaves developed at 5 °C the recovery of photosynthesis as typically seen in WT was strongly inhibited in both antisense lines and this inhibition was associated with a further failure of both antisense lines to cold acclimate. Thus, functional sucrose biosynthesis at low temperature in the over-sps plants reduced the inhibition of photosynthesis, maintained the mobilization of carbohydrates from source leaves to sinks and increased the rate at which freezing tolerance developed. Modification of sucrose metabolism therefore represents an additional approach that will have benefits both for the development of freezing tolerance and over-wintering, and for the supply of exportable carbohydrate to support growth at low temperatures.