Background: Moldova experienced a nationwide mumps outbreak between 2007 and 2008. Single-dose monovalent mumps vaccination at 15 to 18 months was introduced in 1983, replaced by a 2-dose MMR schedule at age 1 and 6 to 7 years in 2002. We investigated the outbreak to quantify its extent, explore the role of primary and secondary vaccine failure, and provide control recommendations. Methods: We analyzed national mumps surveillance and vaccination coverage data to estimate vaccine effectiveness (VE) using the screening method. A retrospective cohort study in 5 educational institutions was conducted to determine age-specific attack rates (ARs) and VE. We compared vaccine strain-specific ARs. Isolation and genotyping of mumps virus strains were performed. Results: Of 31,142 cases reported during October 2007 and July 2008, 80% were in 15-to 24-year-olds. Of cases with information (66%), 92% were vaccinated once, 4% twice. One-dose mumps VE estimates based on surveillance data over 1997-2001 declined from 91% (95% CI: 88%-92%) in 2-year-olds to 72% (70%-74%) in 15-to 19-year-olds. In the cohort study (n = 1589), VE was-40% (-120% to 20%) for 1 dose. For 2 doses it was 62% (-43% to 90%) in 13-to 15-year-olds. ARs were higher in individuals vaccinated with Urabe strains (43%) than with Leningrad-Zagreb strains (14%, P < 0.001). Mumps virus genotype G5 was identified. Conclusions: Low effectiveness of single-dose mumps vaccination was the main cause of the outbreak. Waning immunity may have contributed to this. The risk of mumps in 2-dose vaccinees was low. Other countries in which large population groups have received <2 doses of mumps vaccine may face similar outbreaks.