This study investigated the potential role of MAPK signaling pathways in conjunctivochalasis (CCH). Twenty loose conjunctival biopsy samples from 20 CCH and 15 conjunctival biopsy samples from 15 normal controls (CON) were collected. The conjunctival fibroblasts were cultured in vitro. Immunofluorescence, ELISA, Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were used. Our results showed that the expression of p-ERK, p-JNK, and p-p38 in CCH conjunctiva was significantly higher than that in CON group. The expression of p38 MAPK, JNK, and ERK proteins in CCH fibroblasts was significantly higher than that in CON group. The total expression of MAPK mRNA in CCH fibroblasts was significantly higher than that in CON group. The activated forms of p38 MAPK, JNK, and ERK proteins and mRNAs might up-regulate the expression of MMPs in CCH loose conjunctival tissue and fibroblasts, causing the degradation of collagen fibers and elastic fibers and promoting the occurrence of CCH. Our results deepen the understanding of CCH pathological mechanism.