The c-Jun $NH_2$-terminal kinase (JNK) signaling pathway participates in many physiological functions. In the current study we reported the cloning and characterization of five novel JNK2 transcript variants, which were designated as $JNK2alpha3$, $JNK2alpha4$, $JNK2eta3$, $JNK2gamma1$ and $JNK2gamma2$, respectively. Among them, $JNK2alpha4$ and $JNK2gamma2$ are potential non-coding RNA because they contain pre-mature stop codons. Both $JNK2alpha3$ and $JNK2eta3$ contain an intact kinase domain, and both encode a protein product of 46 kDa, the same as those of $JNK2alpha1$ and $JNK2eta1$. $JNK2gamma1$ contains a disrupted kinase domain and it showed a disable function. When over-expressed in mammalian cells, $JNK2alpha3$ showed higher activity on AP-1 than that of $JNK2eta3$ and $JNK2gamma1$. Furthermore, $JNK2alpha3$ and $JNK2eta3$ showed different levels of substrate phosphorylation, although they both could promote the proliferation of 293T cells. Our results further demonstrate that JNK2 isoforms preferentially target different substrates and may regulate the expression of various target genes.