The rate of improvement of sugar content in sugarcane remains low for decades worldwide. Our previous transcriptome studies provided an atlas of sucrose accumulation-related gene expression but little is known about proteins involved. Here this knowledge gap is addressed by a proteomic analysis of experimentally altered sucrose accumulation in sugarcane. Analysis of stem proteomes of ripener ethephon-treated high- and low-sugar genotypes had identified 2983 proteins of which 139 were significantly differentially expressed (DEPs). These DEPs were found to be involved in sugar metabolism-related processes with 25 of them may have a regulatory role in sucrose accumulation. The key proteins identified include UDP-glucose 6-dehydrogenase associated with amino sugar and nucleotide sugar metabolism; those involved in carbon fixation; and fructokinase, β-D-glucosidase and α-glucan phosphorylase involved in starch and sucrose metabolism. Distinct genotype- and ethephon-dependent DEP expression was evident providing new insights on one of the most intractable sugarcane traits to breeding.