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Anti-thyroid antibody (ATA) positivity affects 1 out of 9 women in childbearing age and presents a significant risk for infertility. Emerging evidence indicates that alterations in the B cell receptor induced calcium (Ca2+) signaling could be key in the development of autoimmunity. We aimed to investigate the Ca2+flux response of B lymphocyte subsets to BCR stimulation in Hashimoto’s thyroiditis and related infertility. We collected peripheral blood samples from ATA+, infertile, euthyroid patients (HIE), hypothyroid, ATA+ patients before (H1) and after levothyroxine treatment (H2), and age-matched healthy controls (HC). All B cell subsets of ATA+, infertile, euthyroid patients showed elevated basal Ca2+level and hyper-responsivity to BCR ligation compared to the other groups, which could reflect altered systemic immune function. The Ca2+flux of hypothyroid patients was similar to healthy controls. The levothyroxine-treated patients had decreased prevalence of CD25+B cells and lower basal Ca2+level compared to pre-treatment. Our results support the role of altered Ca2+flux of B cells in the early phase of thyroid autoimmunity and infertility.