Interleukin-35 (IL-35) is a member of the IL-12 cytokine family and a heterodimeric protein formed by EpsteinBarr virus-induced gene 3 (EBI3) and IL-12p35. Emerging evidence showed that IL-35 is a key player in the regulation of cellular communication, differentiation, and inflammation. To date, no studies on fish IL-35 have been documented. In this work, we first identify two splicing isoforms of EBI3, EBI3a and EBI3b, from grass carp (Ctenopharyngodon idella). EBI3a is composed of 299 amino acid residues and possesses an immunoglobulin-like (Ig-like) domain and a fibronectin type 3 (FN3) domain that is a conservative domain in vertebrate EBI3. However, the EBI3b is composed of 177 amino acid residues and only contains an Ig-like domain. The result of Co-immunoprecipitation suggests that only EBI3a can associate with IL-12p35 to form IL-35 in grass carp. Like the function of IL-35 in human and mouse, recombinant grass carp IL-35 protein could induce the expression of genes EBI3a, IL-12p35, and CD25-like and downregulate the expression of genes CD4-1, CD4-2, IL-17A/F1, and ROR gamma 2. Taken together, these results indicate for the first time that a teleost IL-35 may also have the ability to induce regulatory T (Treg) cells, inhibit effector T (Teff) cell proliferation and restrict the differentiation and function of T helper 17 (Th17) cells in teleost.