Fucosylation plays a critical role in cell-to-cell interactions and disease progression. However, the effects of fucosylation on splenocytes and their interactions with T cells remain unclear. In this study, we aimed to explore the transcriptome profiles of splenocytes deficient in fucosyltransferase (FUT) 1, an enzyme that mediates fucosylation, and investigate their impact on the proliferation and differentiation of T cells. We analyzed and compared the transcriptomes of splenocytes isolated from Fut1 knockout (KO) mice and those from wild-type (WT) mice using RNA-seq. Additionally, we examined the effects of Fut1 KO splenocytes on CD4 T cell proliferation and differentiation, in comparison to WT splenocytes, and elucidated the mechanisms involved. The comparative analysis of transcriptomes between Fut1 KO and WT splenocytes revealed that thrombospondin (THBS)-1, among the genes related to immune response and inflammation, was the most highly downregulated gene in Fut1 KO splenocytes. The reduced expression of THBS1 was further confirmed using qRT-PCR and flow cytometry. In coculture experiments, Fut1 KO splenocytes promoted the proliferation of CD4 T cells and drove their differentiation towards Th1 and Th17 cells, compared to WT splenocytes. Moreover, the levels of IL-2, IFN-γ and IL-17 were increased, while IL-10 was decreased, in T cells cocultured with Fut1 KO splenocytes compared to those with WT splenocytes. These effects of Fut1 KO splenocytes on T cells were reversed when THBS1 was replenished. Taken together, our results demonstrate that splenocytes with Fut1 deficiency promote CD4 T cell proliferation and Th1/Th17 differentiation at least in part through THBS1 downregulation.