The p38 mitogen-activated protein kinase (MAPK) is a member of the MAPK family, which is initially found to be activated by stress stimuli, proinflammatory cytokines, and growth factors. However, its role in the pathogenesis of esophageal squamous cell carcinoma (ESCC) is largely unkown, so we investigate the role of phosphorylated p38 (p-p38) MAPK in ESCC. First of all, in vitro cell line ECa109, SB203580 as selective inhibitor of p38, can suppress the growth of esophageal cancer cell in a dose- and time-dependent way, suggesting that ECa109 cell growth and proliferation was closely associated with p-p38. Using western-blot analysis of fresh 16 paired surgically resected ESCC and matched non-tumor adjacent tissues (NAT), we showed that p-p38 was significantly expressed higher in NAT compared to ESCC. Moreover, expressions of p-p38 were further confirmed by 162 paired of formalin-fixed paraffin-embedded (FFPE) ESCC and NAT by immunohistochemistry, the same trend result was obtained through statistical analysis that there was increased expression of p-p38 in NAT as compared with ESCC (P < 0.01), and expression of p-p38 was not significantly associated with lymph nodes metastasis (P > 0.05) and ESCC differentiation degree (P > 0.05). Taken together, all the results we obtained demonstrated that p-p38 plays a key role in the malignant transformation of ESCC.