Glandular secreting trichomes on the surface of tomato plants and many of its relatives in the Solanaceae produce a mixture of O -acyl sugars that contribute to insect resistance. The majority of acyl sucroses produced by the cultivated tomato ( Solanum lycopersicum ) contain three or four short chain aliphatic acyl esters, and tetra-acyl sucroses have an acetyl group as one of the acyl chains. We previously reported overlapping S. lycopersicum × Solanum pennellii introgression lines (ILs) that fail to accumulate high levels of acetylated tetra-acyl sucroses. A survey of the annotated genes in this region of cultivated tomato chromosome 1 revealed three candidate acyltransferases that were tested for function using virus-induced gene silencing. A member of the BAHD family of acyltransferases (Solyc01g105580, SlAT2 ) was shown to encode an acetyl-CoA–dependent acyltransferase enzyme capable of acyl sucrose acetylation in vitro. RNAi suppression of SlAT2 in transgenic S. lycopersicum cv. M82 resulted in reduced acyl sugar acetylation, whereas expression of the functional S. lycopersicum allele of SlAT2 in the triacyl sucrose producing IL1-3 restored the ability of the IL to synthesize acetylated tetra-acyl sugars. Transgenic plants with the SlAT2 promoter driving GFP expression showed fluorescence in tips cells of long, slender trichomes that is consistent with acyl sugar acetylation occurring in these cells.