Fragrance in table grapes and a persistent and complex aroma in wine are both sought after by the modern consumer. In particular, the floral flavor typical of Muscat varieties, due to high levels of monoterpenoids (geraniol, linalool and nerol), is highly distinct and has been greatly appreciated since ancient times. Muscat flavor determination in grapevine (Vitis vinifera L.) has up to now been studied by evaluating monoterpenoid quantity through QTL analysis. These studies have revealed co-localization of 1-deoxyxylulose-5-phosphate synthase (VvDXS) with the major QTL positioned on chromosome 5. The aim of the present study was to assess the connection between the positional candidate gene VvDXS and muscat flavor by investigating the nucleotide diversity of VvDXS ORF and its expression profiles in the grape berry. Association between VvDXS polymorphisms and muscat flavor was evaluated through a structured association analysis. A putative causal SNP responsible for a predicted non-neutral substitution was found to be significantly associated with muscat-flavored varieties. Moreover, muscat-like aromatic mutants displayed unique heterozygous non-synonymous mutations near the mutated site of Muscat genotypes. The relationship between the transcription profile of VvDXS alleles and monoterpenoid accumulation was investigated over an extended period of berry development. It is possible that a particular trend in expression rather than the level of expression ratio affects monoterpenoid accumulation in Moscato Bianco. In vivo experiments using transgenic tobacco and in vitro enzymatic assays suggest that the putative causal SNP raises monoterpenoid accumulation by changing the 3D protein structure and by increasing VvDXS activity in Muscat enzyme form.