Renal fibrosis is a histological manifestation that occurs in almost every type of chronic kidney disease. Histone variant H3.3 and its chaperone, histone cell cycle regulation defective homolog A (HIRA), serve as epigenetic marks that regulate transcriptional activity. In this study, we assessed the roles of histone H3.3 and HIRA in unilateral ureteral-obstruction (UUO) mice. In UUO mice, the levels of histone H3.3 and HIRA were significantly upregulated in the kidneys. These upregulated levels were decreased by a TGF-β1 neutralizing antibody. TGF-β1 induced histone H3.3 and HIRA expression in vitro via a Smad3-dependent pathway in normal rat kidney (NRK)-52E cells. Additionally, knockdown of HIRA expression decreased histone H3.3 expression and fibrogenesis in NRK-52E cells after TGF-β1 stimulation. Chromatin immunoprecipitation analysis revealed that promoters of fibrosis-related genes were immunoprecipitated with both histone H3.3 and HIRA in NRK-52E cells. Lastly, in human kidney biopsies from patients diagnosed with IgA nephropathy, histone H3.3 and HIRA immunostaining correlated positively with areas of fibrosis and estimated glomerular filtration rate. In conclusion, TGF-β1 induces expression of histone H3.3 and HIRA, which regulates expression of fibrosis-related genes.