Polo-like kinase 1 (Plk1) is a critical regulator of many stages of mitosis; increasing evidence indicates that Plk1 overexpression correlates with poor clinical outcome, yet its mechanism of regulation remains unknown. Hence, a detailed evaluation was undertaken of Plk1 expression in human nasopharyngeal cancer (NPC), the cellular effects of targeting Plk1 using siRNA in combination with ionizing radiation (RT) and potential upstream microRNAs (miRs) that might regulate Plk1 expression. Using immunohistochemistry, Plk1 was observed to be overexpressed in 28 of 40 (70%) primary NPC biopsies, which in turn was associated with a higher likelihood of recurrence (p = 0.018). SiPlk1 significantly inhibited Plk1 mRNA and protein expression, and decreased Cdc25c levels in NPC cell lines. This depletion resulted in cytotoxicity of C666-1 cells, enhanced by the addition of RT, mediated by G2/M arrest, increased DNA double-strand breaks, apoptosis, and caspase activation. Immunofluorescence demonstrated that the G2/M arrest was associated with aberrant spindle formation, leading to mitotic arrest. In vivo, transfection of C666-1 cells and systemic delivery of siPlk1 decreased tumour growth. MicroRNA-100 (miR-100) was predicted to target Plk1 mRNA, which was indeed underexpressed in C666-1 cells, inversely correlating with Plk1 expression. Using luciferase constructs containing the 3'-UTR of Plk1 sequence, we document that miR-100 can directly target Plk1. Hence, our data demonstrate for the first time that underexpressed miR-100 leads to Plk1 overexpression, which in turn contributes to NPC progression. Targeting Plk1 will cause mitotic catastrophe, with significant cytotoxicity both in vitro and in vivo, underscoring the important therapeutic opportunity of Plk1 in NPC.