Tuberculosis (TB) is a leading cause of global mortality due to infectious diseases. Expression of cyclooxygenase-2 (COX-2) acts as an important influencing factor favoring bacillary survival during TB infection. In this study, we investigated the Mycobacterium tuberculosis proteins recognized by sera from TB patient collected before and after anti-TB therapy by dynamic immunoproteomics and identified a novel immune-regulating protein 3-hydroxyacyl-l-thioester dehydratase Y (HtdY), which could induce COX-2 expression in mouse macrophages. Signaling perturbation data showed that the activation of p38, ERK 1/2 and JNK 1/2 MAPK as well as NF-κB played critical role in this immune response. Taken together, our findings indicated that mycobacterial HtdY might contribute to the persistence of the TB infection by inducing COX-2 expression through MAPK-NF-κB signaling pathway.