The modern day western diet has led to an increase of n-6 polyunsaturated fatty acid (n-6 PUFA) intake and is considered an environmental factor resulting in negative health outcomes in different disease settings. In relation to the immune system, it is not well understood how changes in dietary factors like n-6 PUFAs can influence immune responses, for example during autoimmune disease. PUFAs have a variety of roles within cells such as being used as an energy source, incorporation in cell membranes, and conversion into lipid mediators that influence cell signaling and gene expression. We initially sought to understand how n-6 PUFAs influence the phenotype and function of T cell subsets from the Type 1 Diabetes mouse model (NOD) and non-diabetic control mouse models. By treating splenocytes from these mouse models in vitro with n-6 PUFA linoleic acid we discovered by flow cytometry a dose response inhibition of anti-inflammatory interleukin 10 (IL-10) in both CD4+ and CD8+ T cell subsets. While the inhibition was not strain-specific, further studies by gene expression analysis displayed a decrease of IL-10 mRNA expression and of transcription factors involved in regulation of IL-10 (c-maf, IRF4, and Bhlhe40) after linoleic acid treatment. These results demonstrate that dietary n-6 PUFAs regulate T cell cytokine production and can influence the balance of pro- and anti-inflammatory cytokines. Further studies will provide insight into the regulation of T cell subsets by fatty acids present in the organismal environment, with potential implications for the development of new therapeutics to modulate autoimmune responses.