As a multifunctional cytokine, macrophage migration inhibitory factor (MIF) is associated with inflammation and tumorigenesis; however, the role of MIF in cervical adenocarcinoma (ADC) is not fully understood. In this study, we aimed to examine the expression of MIF in ADC and explore the mechanism of MIF in ADC progression. MIF expression was positively related to ADC clinicopathological features of carcinoma diameter and lymph node metastasis. MIF knockdown induced cell cycle arrest of G1/S transition in ADC cells, upregulation of the expressions of p21 and p27, and downregulation of the expressions of Cdk4, CyclinD2, and CyclinE2. In MIF knockdown cells, the expressions of proapoptotic proteins of Bax, caspase-3, cleaved caspase-3, and cleaved-PARP were upregulated, and the expressions of antiapoptotic proteins of Bcl-2, pAkt, and p53 were downregulated. It indicated that MIF knockdown inhibited cell proliferation and induced apoptosis in ADC cells. MIF might be a novel molecular marker in diagnosis and therapy of ADC.