Neural retina leucine zipper (Nrl), a key basic motif leucine zipper (bZIP) transcription factor, modulates rod photoreceptor differentiation by activating rod-specific target genes. In searching for factors that might couple with Nrl to modulate its transcriptional activity through posttranslational modification, we observed the novel interactions of Nrl with c-Jun N-terminal kinase 1 (JNK1) and HIV Tat-interacting protein 60 (Tip60). JNK1 directly interacted with and phosphorylated Nrl at serine 50, which enhanced Nrl transcriptional activity on the rhodopsin and Ppp2r5c promoters. Use of an inactive JNK1 mutant or treatment with a JNK inhibitor (SP600125) significantly reduced JNK1-mediated phosphorylation and transcriptional activity of Nrl in cultured retinal explants. We also found that Nrl activated rhodopsin and Ppp2r5c transcription by recruiting Tip60 to promote histone H3/H4 acetylation. The binding affinity of phospho-Nrl for Tip60 was significantly greater than that of the unphosphorylated Nrl. Thus, the histone acetyltransferase-containing Tip60 behaved as a coactivator in the Nrl-dependent transcriptional regulation of the rhodopsin and Ppp2r5c genes in the developing mouse retina. A transcriptional network of interactive proteins, including Nrl, JNK1, and Tip60, may be required to precisely control spatiotemporal photoreceptor-specific gene expression during retinal development.