The presence of senescent cells is associated with renal fibrosis. This study aims to investigate the effect of albumin-induced premature senescence on tubulointerstitial fibrosis and its possible mechanism . Different concentrations of bovine serum albumim (BSA) with or without si-p21 are used to stimulate HK-2 cells for 72 h, and SA-β-gal activity, senescence-associated secretory phenotypes (SASPs), LaminB1 are used as markers of senescence. Immunofluorescence staining is performed to characterize the G2/M phase arrest between the control and BSA groups. Alterations in the DNA damage marker γ-H2AX, fibrogenesis, and associated proteins at the G2/M phase, such as p21, p-CDC25C and p-CDK1, are evaluated. Compared with those in the control group, the SA-β-gal activity, SASP, and γ-H2AX levels are increased in the BSA group, while the level of LaminB1 is decreased. Meanwhile, HK-2 cells blocked at the G2/M phase are significantly increased under the stimulation of BSA, and the levels of p21, p-CDC25C and p-CDK1, as well as fibrogenesis are also increased. When p21 expression is inhibited, the levels of p-CDC25C and p-CDK1 are decreased and the G2/M phase arrest is improved, which decreases the production of fibrogenesis. In conclusion, BSA induces renal tubular epithelial cell premature senescence, which regulates the G2/M phase through the CDC25C/CDK1 pathway, leading to tubulointerstitial fibrosis.