CD81 is involved in leukocyte migration and cytokine induction. Previous work found that anti-CD81 monoclonal antibodies (mAbs) showed therapeutic potential for several immune diseases via inhibiting leukocyte migration. Although the suppression of cell migration is a promising approach for treating immune diseases, some anti-CD81 mAbs can induce cytokine production, which may exacerbate disease. To obtain new anti-human CD81 mAbs that inhibited migration in the absence of cytokine production enhancement activity, we screened a human single chain variable fragment by phage library. One of the new anti-CD81 mAbs isolated, DSP-8250, had equivalent inhibitory cell migration activity with the established anti-CD81 mAb 5A6, but it lacked cytokine induction activity. These mAbs recognized different epitopes on CD81. mAb 5A6, which had inhibitory activity on T-cell migration and increased cytokine production, bound to three residues, Ser179, Asn180 and Phe186 of CD81. In contrast, DSP-8250, which had inhibitory activity on T-cell migration but no cytokine enhancement activity, bound to four residues, His151, Ala164, Ser168 and Asn172 of CD81 as a unique epitope. These results indicate that the set of His151, Ala164, Ser168 and Asn172 forms a novel epitope that might make the application of anti-CD81 mAb therapeutically useful.