Interleukin (IL)‐1β has been shown to induce matrix metalloproteinase (MMP)‐9 expression through mitogen‐activated protein kinases, including JNK, in rat brain astrocyte‐1 (RBA‐1) cells. However, little is known about whether JNK activated by Ca²⁺‐dependent CaMKII is associated with MMP‐9 expression induced by IL‐1β. Here, we report that the Ca²⁺/CaMKII/JNK/c‐Jun participates in the MMP‐9 expression induced by IL‐1β. Zymographic, Western blotting, and RT‐PCR analyses showed that IL‐1β‐induced expression of MMP‐9 mRNA and protein was attenuated by Ca²⁺ chelator (BAPTA), and the inhibitors of ER Ca²⁺‐ATPase (thapsigargin), CaMKII (KN‐62), and JNK1/2 (SP600125). IL‐1β also stimulated phosphorylation of CaMKII and JNK1/2, and increase in intracellular Ca²⁺ ([Ca²⁺]_i), which were inhibited by pretreatment with BAPTA, thapsigargin (TG), KN‐62, or SP600125. Furthermore, the upregulation of MMP‐9 protein was blocked by transfection with c‐Jun or CaMKII short hairpin RNA (shRNA). We further confirmed that IL‐1β stimulated c‐Jun associated with AP‐1‐binding sites within MMP‐9 promoter (−87 to −80 bp and −511 to −497 bp) by immunoprecipitation and chromatin immunoprecipitation (ChIP)‐PCR assays. The activation and recruitment of c‐Jun to MMP‐9 promoter were inhibited by pretreatment with BAPTA, TG, KN‐62, or SP600125. Moreover, IL‐1β‐induced MMP‐9 gene transcription by AP‐1 was confirmed by transfection with a MMP‐9 promoter‐luciferase reporter plasmid with a distal AP‐1‐binding site (−511 to −497 bp) adjacent to an Ets‐binding site‐mutation (mt‐AP1/Ets‐MMP‐9). These results demonstrated that in RBA‐1 cells, JNK/c‐Jun activation was mediated through a Ca²⁺‐dependent CaMKII pathway that promoted transcription factor c‐Jun/AP‐1 recruitment and eventually led to increase in MMP‐9 expression by IL‐1β. © 2009 Wiley‐Liss, Inc.