PIRIN2 (PRN2) was earlier reported to suppress S-type lignin accumulation of xylem vessels of Arabidopsis (Arabidopsis thaliana). In the present study we report yeast-two hybrid results supporting the interaction of PRN2 with HISTONE MONOUBIQUITINATION2 (HUB2) in Arabidopsis. HUB2 has been previously implicated in several plant developmental processes, but not in lignification. Interaction between PRN2 and HUB2 was verified by β-galactosidase enzymatic and co-immunoprecipitation assays. HUB2 promoted the deposition of S-type lignin in the secondary cell walls of both stem and hypocotyl tissues, as analysed by pyrolysis‐gas chromatography/mass spectrometry. Chemical fingerprinting of individual xylem vessel cell walls by Raman and Fourier Transform Infrared microspectroscopy supported the function of HUB2 in lignin deposition. These results, together with a genetic analyses of the hub2 prn2 double mutant, support antagonistic function of PRN2 and HUB2 in deposition of S-type lignin. Transcriptome analyses indicated the opposite regulation of the S-type lignin biosynthetic FERULATE-5-HYDROXYLASE1 by PRN2 and HUB2 as the underlying mechanism. PRN2 and HUB2 promoter activities co-localised in cells neighbouring the xylem vessel elements, suggesting that the S-type lignin promoting function of HUB2 is antagonized by PRN2 for the benefit of the G-type lignin enrichment of the neighbouring xylem vessel elements.