Animal cells adapt to the stiffness of their environment through mechanotransduction, a process in which mechanical signals are converted into biochemical responses, influencing key cellular processes such as growth and differentiation. We identified ubiquitin-conjugating enzymes E2 A and B (UBE2A/B) as mechanosensitive proteins that translocate between the nucleus and cytoplasm depending on force and substrate stiffness. Here, we hypothesized that UBE2A/B nuclear translocation on stiff substrates triggers gene expression via UBE2A/B-mediated ubiquitination of histone H2B lysine 120 (H2BK120). Chromatin immunoprecipitation sequencing (ChIP-seq) revealed distinct DNA fragments bound to monoubiquitinated H2B in cells cultured on soft (0.2 kPa) versus stiff (64 kPa) substrates. We identified 2245 gene regions binding to ubiquitinated histones on stiff substrates and 294 on soft substrates and further integrated RNA-seq and UBE2A/B knockdown data to pinpoint 179 stiff-specific and 18 soft-specific genes. Among these, filamin C (FLNC), leucine zipper protein 1 (LUZP1), and glutamate-rich WD repeat-containing protein 1 (GRWD1) showed higher expression on stiff substrates, with GRWD1 known for its role in cancer progression through cell cycle and gene regulation. These findings highlight how substrate stiffness modulates gene expression via UBE2A/B-mediated H2B ubiquitination.