Background: More than 40% of nasopharyngeal carcinoma (NPC) come from China, and the incidence rate in South China is extremely high. Poorly differential nasopharyngeal squamous carcinoma (PDNSC) is the most common pathological type, while the detailed molecular mechanisms remain unclear. Objectives: This study aimed to explore the role of MYL9 in the carcinogenesis, progression and chemoresistance of PDNSC, as well as the specific molecular biological mechanisms of pathogenesis and paclitaxel resistance, and to provide a new theoretical basis of targeted drugs for PDNSC. Methods: In this study, Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot were performed to detect the mRNA and protein expression of MYL9 in PDNSC tissues and cell lines (HONE-1 and SUNE-1), respectively. Moreover, MTT and EdU assays were performed to detect the proliferation function of MYL9 in PDNSC cells; Transwell and Boyden assays were performed to detect the migration and invasion function of MYL9; The IC50 value of paclitaxel was performed to detect the effect of MYL9 on drug sensitivity in PDNSC cells. In addition, Western blot assay was performed to detect the mechanism of MYL9 in PDNSC cells. Results: The results showed that MYL9 mRNA was much higher in PDNSC tissues compared with that in paratumor tissues, and MYL9 knockdown suppressed the proliferation, migration and invasion, while reducing the IC50 value of paclitaxel, in PDNSC cells. Mechanistic study showed that MYL9 knockdown inhibits the epithelial-mesenchymal transition (EMT) signals and downstream chemoresistant factors including ABCG2 and ABCB1. Conclusion: MYL9 is upregulated in PDNSC, and it promotes PDNSC carcinogenesis, progression and paclitaxel resistance via regulating EMT signals and downstream chemoresistant factors, and it may be used as a useful patent of therapeutic target in PDNSC.