Two anti-MSLN VHH mAb clones (MT1A1 and MT3C2), targeting the extracellular domain of human MSLN, recognize the region (296–390) at the N-terminal of MSLN on the cell surface and relying on their conformation-dependent recognition. MT1A1 and MT3C2 significantly inhibited the MSLN-CA125 interaction. Overall, our results suggest that MT1A1 and MT3C2 are promising anticancer agents.