B cell-mediated regulatory function is instrumental to the maintenance of tolerance, but may also contribute to immune dysfunction during infectious diseases and malignancies. In this study, we investigated a subset of B cells characterized by PD-1 expression. Data showed that these PD-1⁺ B cells were rare in peripheral blood, but were significantly upregulated in differentiated thyroid tumors. The PD-1⁺ B cells also expressed significantly higher level of PD-L1. Continuous, but not short-term, anti-Ig/CD40 L stimulation could upregulate the expression of PD-1 and PD-L1 in B cells. In in vitro experiments, PD-1⁺ B cells significantly suppressed the proliferation of CD4⁺ and CD8⁺ T cells and reduced their viability upon CD3/CD28 stimulation, thus suggesting that these PD-1⁺ B cells presented regulatory functions. However, unlike other IL-10-secreting Breg cell subsets, the PD-1⁺ B cells did not express high level of IL-10. Instead, it seemed that PD-L1 was instrumental to the suppressive effects mediated by PD-1⁺ B cells, since the blockade of PD-L1 significantly increased the proliferation and viability of T cells in the coculture. Interestingly, compared to untreated patients with differentiated thyroid tumor, the thyroidectomy and ¹³¹I-treated patients presented significantly lower frequencies of PD-1⁺ B cells. Together, our investigation demonstrated that the PD-1⁺ B cells possessed regulatory capacity toward T cell responses, and although rare in peripheral blood, they were significantly enriched in thyroid tumors.