Myosin light chains (MLC) serve important regulatory functions in a wide range of cellular and physiological processes. Recent research found that MLC are also chromatin-associated nuclear proteins which regulate gene transcription. In this study, the MLC member myosin regulatory light chain 5 (MYL5) expression was upregulated in late stage cervical cancer patients, positively correlated with pelvic lymph node metastasis, and identified as a poor survival indicator. MYL5 overexpression promoted metastasis in cervical cancer in vitro and in vivo models, whereas MYL5 silencing had the converse effect. We demonstrated a bidirectional regulation between MYL5 and hypoxia inducible factor-1 alpha (HIF-1 alpha). HIF-1 alpha activates MYL5 via binding to the hypoxia response element (HRE) in the promoter of MYL5, and MYL5 could sustain HIF-1 alpha expression by tethering to recognition sequence AGCTCC in the HIF-1 alpha promoter region. Clinical data confirmed a positive correlation between MYL5 and HIF-1 alpha. In summary, our data show that MYL5 may act as a prognosis predictive factor in cervical carcinoma, and strategies that inhibit the interaction of MYL5 and HIF-1 alpha may benefit the cervical carcinoma patients with metastasis.