Anti-endothelial cell antibodies (AECAs) have been identified in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and in patients with idiopathic pulmonary arterial hypertension (iPAH). However, their target antigens remain poorly identified. Sera from 24 patients with SSc without PAH, 20 patients with SSc with PAH, 30 with iPAH and 12 healthy controls were collected. Target antigens were identified by two-dimensional electrophoresis and immunoblotting in protein extracts of human umbilical vein endothelial cells. Targeted antigens were identified by mass spectrometry. Serum immunoglobulin G from patients with SSc with or without PAH and patients with iPAH specifically recognised 110, 82 and 37 protein spots, respectively. Among others, target antigens of AECAs included lamin A/C, tubulin β-chain and vinculin. One-dimension immunoblotting experiments confirmed the identification of lamin A/C and tubulin β-chain. In conclusion, our results confirm the presence of AECA in patients with systemic sclerosis with and without pulmonary arterial hypertension and in those with idiopathic pulmonary arterial hypertension, and provide evidence for the identification of target antigens of these autoantibodies including lamin A/C and tubulin β-chain.