Proinsulin C-peptide has been reported to have some biological activities and to be possibly involved in the development of diabetic microangiopathy. In the present study, we examined the effects of C-peptide on the mitogen-activated protein kinase pathway in LEII mouse lung capillary endothelial cells. Stimulation of the cells with C-peptide increased both p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK1/2) activities and activity-related site-specific phosphorylation of the respective kinases in a concentration-dependent manner, but failed to activate c-Jun N-terminal kinase. Stimulation of the cells with C-peptide also induced site-specific phosphorylation of cAMP response element (CRE)-binding protein (CREB)/activating transcription factor 1 (ATF1), and thereby binding of these transcription factors to CRE. Among three CREB kinases tested, phosphorylation of mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2) was induced after stimulation with C-peptide. The phosphorylation of CREB, ATF1 and MAPKAP-K2 were inhibited by SB203580, a p38MAPK inhibitor, but not by PD98059, an ERK kinase inhibitor. These results indicate that C-peptide activates p38MAPK followed by MAPKAP-K2 to enhance DNA-CREB/ATF1 interactions.