During austral winter, the southern and eastern coastlines of South Africa witness one of the largest animal migrations on the planet, the KwaZulu-Natal sardine run. Hundreds of millions of temperate sardines, Sardinops sagax, form large shoals that migrate north-east towards the subtropical Indian Ocean. Recent studies have highlighted the role that genetic and environmental factors play in sardine run formation. In the present study, we used massively parallel sequencing to assemble and annotate the first reference transcriptome from the liver cells of South African sardines, and to investigate the functional content and transcriptomic diversity. A total of 1,310,530 transcripts with an N50 of 1578 bp were assembled de novo. Several genes and core biochemical pathways that modulate energy production, energy storage, digestion, secretory processes, immune responses, signaling, regulatory processes, and detoxification were identified. The functional content of the liver transcriptome from six individuals that participated in the 2019 sardine run demonstrated heterogeneous levels of variation. Data presented in the current study provide new insights into the complex function of the liver transcriptome in South African sardines.