CD4+ T helper cells are involved in multiple biological processes ranging from pathogen clearance to immune tolerance. Differentiation of CD4+ T cells into varied subsets is dependent on transcription factors, cytokines, micronutrients, and epigenetic modifications. Here, we report the molecular mechanisms imparted by the homeobox transcription factor HOXB4 in modulating Th2 and Th9 cell differentiation. We found that Hoxb4 induction in T helper cells was TGF-β-dependent. In Th9 cells, HOXB4 overexpression significantly increased IL-9 secretion, while in Th2 cells, HOXB4 could alter only IL-10 secretion among other type 2 cytokines. Promoter activity analyses revealed that HOXB4 was able to transactivate the Il9 and Il10 proximal promoters, while the Il4 and Il13 promoters were differentially modulated. We observed that HOXB4 could physically interact with the transcription factor PU.1 but not GATA3, and directly bind PU.1 response element on the DNA in Th9 cells. The recruitment of HOXB4 was found to be significantly increased in regulatory regions including cECR and CNS1 (Il9 promoter). Collectively, our findings suggest that HOXB4 works downstream of the TGF-β signalling pathway, associates with PU.1, binds the regulatory regions, and distinctly regulates both Th2 and Th9 cell differentiation.