Heparin-induced thrombocytopenia (HIT) related to fondaparinux has been rarely reported, although the ability of fondaparinux to cross-react with heparin antibodies has been often a subject of debate. A patient previously exposed to unfractionated heparin and low-molecular-weight heparin (LMWH) was diagnosed with HIT. During treatment with fondaparinux for 5 consecutive days, his thrombocytopenia significantly deteriorated. A functional platelet activation test in vitro showed clear platelet activation after serum exposure with fondaparinux. After discontinuation of fondaparinux, the platelet count was rapidly reestablished. Fondaparinux cross-reacted with heparin antibodies in this case of HIT, resulting in a deterioration of thrombocytopenia. The implication of this drug in HIT was observed clinically and demonstrated in vitro using a platelet activation test.