Interleukin 17A-producing T helper cells (Th17) are effector memory CD4+ T cells that are crucial to adaptive immunity to extracellular bacteria. The activities of these cells in the bovine species are not yet defined for want of straightforward cultivation and isolation procedures. We have developed a method to cultivate, expand, sort and maintain in culture bovine Th17 cells from circulating CD4+ T cells of adult cows. Using polyclonal stimulation (antibodies to CD3 and CD28), we expanded positive CD4+ IL-17A+ T (Th17) cells in a cell culture medium without serum supplemented with TGF-β1 and IL-6. We used IL-2 to expand the cells, which were characterized by intracellular labeling for IL-17A and IFN-γ. Then, we isolated populations of CD4+ T cells producing IL-17A, IFN-γ or both by labeling surface IL-17A with either a complex of biotinylated anti-CD45 antibodies-streptavidin-biotinylated anti-IL-17A antibodies or by direct surface labeling with antibodies to IL-17A, followed by flow cytometry cell sorting. The percentages of surface-labeled IL-17A-secreting cells were quite similar to the percentages of intracellular-labeled IL-17A-producing cells of the same cultures, and the two labeling procedures of live cells yielded similar results. The sorted IL-17A+ cells were restimulated and expanded. After expansion, 80% of the isolated Th17 cells were positive for IL-17A intracellular labeling. The sorted IL-17A+ cells can be frozen, stored and expanded again. The sorted Th17 cells secreted much more IL-17A and IL-17F than did CD4+ IL-17- cells. Notably, most of Th17 cells secreted IFN-γ, although in lower amounts than did CD4+ IL-17- cells. Sorted cells were characterized by transcriptomic profiling. Genes coding for Th17 signature cytokines (IL-17A, IL-17F, IL-26) and transcription factors (RORγt, RORa) were overexpressed in Th17 cells. The techniques developed will make it possible to investigate the phenotypic and functional profiles of bovine Th17 cells along with their stability or plasticity.