Cisplatin resistance is a major obstacle in the treatment of lung adenocarcinoma (LAD), and its mechanism has not been fully elucidated. Here, we report that miR-326 is downregulated in cisplatin-resistant A549/CDDP cells compared with parental A549 cells. Overexpression of miR-326 reversed cisplatin chemoresistance of LAD cells in vitro and in vivo. Moreover, we identified the specificity protein 1 (SP1) gene as a novel direct target of miR-326. Knockdown of SP1 revealed similar effects as that of ectopic miR-326 expression. Decreased miR-326 expression was also detected in tumor tissues sampled from LAD patients treated with cisplatin-based chemotherapy and was proved to be correlated with high expression of SP1 and decreased sensitivity to cisplatin. Furthermore, we show that the long noncoding RNA HOTAIR repression reverses chemoresistance of LAD cells partially through modulation of miR-326/SP1 pathway. In summary, we unveil a branch of the HOTAIR/miR-326/SP1 pathway that regulates chemoresistance of LAD cells.