BACKGROUND The upregulation of serpin family B member 5 (SERPINB5) has been linked to the progression of rectal cancer. However, the specific roles and underlying mechanisms of SERPINB5 in rectal cancer are not fully understood. AIM To investigate the roles and mechanisms of SERPINB5 in rectal cancer. METHODS SERPINB5 protein level in rectal cancer tissues and cell lines was measured through western blot analysis. SW480 cells were transfected with pcDNA-SERPINB5 or short-hairpin RNA targeting SERPINB5 (sh-SERPINB5). Cell proliferation, invasion, and apoptosis were then evaluated. The interaction between SERPINB5 and heat shock protein 90 alpha class A member 1 (HSP90AA1) was confirmed through a co-immunoprecipitation assay. Subsequently, pcDNA-HSP90AA1 or sh-HSP90AA1 was transfected into SW480 cells, and cell progression was then detected. Moreover, rescue experiments were used to investigate the effect of the SERPINB5/HSP90AA1 axis on rectal cancer progression. Additionally, sh-SERPINB5-transfected SW480 cells were implanted into nude mice, and xenograft tumor growth was then evaluated. RESULTS SERPINB5 was prominently upregulated in rectal cancer tissues and cells. SERPINB5 overexpression increased SW480 cell proliferation and invasion while reducing apoptosis. In contrast, SERPINB5 knockdown had the opposite effects. Moreover, SERPINB5 could interact with HSP90AA1 and promote HSP90AA1 expression in SW480 cells. HSP90AA1 overexpression facilitated SW480 cell proliferation and invasion and restrained apoptosis. By contrast, HSP90AA1 knockdown suppressed cell progression. The upregulation of HSP90AA1 reversed the SERPINB5 silencing-mediated inhibition of SW480 cell progression. Additionally, SERPINB5 knockdown retarded the growth of rectal cancer tumors in vivo. CONCLUSION SERPINB5 knockdown inhibited rectal cancer cell proliferation and invasion and retarded xenograft tumor growth by inhibiting HSP90AA1 expression.