Hsf4b, a key regulator of postnatal lens development, is subjected to posttranslational modifications including phosphorylation. However, the phosphorylation sites in Hsf4b and their biological effects on the transcription activity of Hsf4b are poorly understood. Here we examined 17 potential phosphorylation residues in Hsf4b with alanine-scanning assays and found that a T472A mutation diminished Hsf4b-mediated expression of Hsp25 and alphaB-crystallin. In contrast, the phosphomimetic mutation of T472D enhanced their expression. Further investigation demonstrated that Hsf4b could interact with nuclear-transporter importin beta-1 and Hsc70 via amino acids 246-320 and 320-493, respectively. T472A mutation reduced Hsf4bs interaction with importin beta-1, while enhancing its interaction with Hsc7O, resulting in Hsf4b cytosolic re-localization, protein instability and transcription activity attenuation. At the upstream, MEK6 was found to interact with Hsf4b and enhance Hsf4b's nuclear translocation and transcription activity, probably by phosphorylation at sites such as T472. Taken together, our results suggest that phosphotylation of Hsf4b at T472 by protein kinases such as MEI(6 regulates Hsf4b interaction with the importin V I -Hsc7O complex, resulting in blockade of its nuclear translocation and transcriptional activity of Hsf4b.