Cervical cancer is the second most common malignant cancer in females. Recent findings indicate that LncRNA-HOTAIR played a crucial role in tumor progression. In our present study, we aimed to explore the regulating role of HOTAIR in the progression of cervical cancer. The expression of HOTAIR was found up-regulated in cervical cancer tissues and cell lines (Hela, CasKi, Me180 and C-33A) compared with the normal tissues and normal cervical cell line (Ect1/E6E7). To examine the function of HOTAIR, gene knockdown (KD) was performed using HOTAIR short hairpin RNAs (shRNAs). HOTAIR shRNA significantly suppressed cells proliferation and migration in Hela cells. Besides that, the targeting relationship between HOTAIR and miR-326 was firstly revealed by bioinformatics prediction. Simultaneously, suppressed expression of miR-326 was detected in tumor tissues and cell lines compared with the control. Then, suppressed expression level of miR-326 was elevated by adding miR-326 mimic in cervical cancer cells transfected with LncR-HOTAIR. Similarly, increased expression level of miR-326 was reduced by adding miR-326 inhibitor in cervical cancer cells transfected with HOTAIR shRNA. The targeting relationship between HOTAIR and miR-326 was further been confirmed through the luciferase report assay. Moreover, there existed a negative relationship between the expression of HOTAIR and miR-326. In addition, enhanced cell proliferation and migration abilities were suppressed by HOTAIR shRNA in cells transfected with miR-326 inhibitor. Finally, the in vivo experiment revealed that tumor growth and metastasis could also be inhibited by HOTAIR shRNA. Our present study elucidated the regulating role of HOTAIR/miR-326 axis in cervical cancer progression and provided a new potential therapeutic strategy for cervical cancer.