Phenotypic plasticity is an important driver of species resilience. Often mediated by epigenetic changes, phenotypic plasticity enables individual genotypes to express variable phenotypes in response to environmental change. Barramundi (Lates calcarifer) is a protandrous (male-first) sequential hermaphrodite that exhibit plasticity in length-at-sex change between geographic regions. This plasticity is likely to be mediated by changes in DNA methylation (DNAm), a well-studied epigenetic modification. However, region-specific relationships between length, sex and DNAm in sequential hermaphrodites were previously unreported. To investigate these relationships, here we compare DNAm in four conserved vertebrate sex-determining genes in male and female barramundi of differing lengths from three regions of northern Australia. Despite a strong association between increasing length and male-to-female sex change, the data reveal that DNAm becomes more sex-specific (rather than more female-specific) with length. Significant differences in DNAm between males and females of similar lengths suggest that female-specific DNAm arises rapidly during sex change, rather than gradually with growth. The findings also reveal that region-specific differences in length-at-sex change are accompanied by differences in DNAm, and were concurrent with variability in remotely sensed sea temperature and salinity. Together, these findings provide the first in situ evidence for epigenetically and environmentally mediated sex change in a protandrous hermaphrodite, and offer significant insight into the molecular and ecological processes governing the marked and unique plasticity of sex in fish.