Objective Psoriasis is an immune-mediated inflammatory disease. Despite advances in the study of its pathogenesis, the exact development mechanism of psoriasis remains to be fully elucidated. Hyperproliferative epidermis plays a crucial role in psoriasis. This study aimed to investigate the effects of interleukin-36β (IL-36β) on keratinocyte dysfunction in vitro. Methods Human keratinocyte cell lines, HaCaT cells, were treated with 0 (control), 50 or 100 ng/ml IL-36β respectively for 24 h. Cell viability was determined with a cell counting kit-8 assay. Flow cytometry was used to assess the effects of IL-36β on apoptosis and cell cycle distribution. Expressions of the differentiation markers, such as keratin 10 and involucrin, were evaluated by quantitative real-time polymerase chain reaction (RT-qPCR). Expressions of the inflammatory cytokines, IL-1β and IL-6 were tested by ELISA. Results CCK8 assay showed the survival rate had no significant difference between the control and treated group (P > 0.05). Flow cytometry analysis showed cell cycle arrest at S phase in the IL-36β-treated groups compared with the control group (P < 0.05). RT-qPCR verified the decreased mRNA expressions of keratin 10 and involucrin in the IL-36β-treated groups compared with the negative control (P < 0.01). ELISA showed 100 ng/ml IL-36β enhanced levels of IL-1β and IL-6 in culture supernatants of HaCaT cells compared with the negative control (P < 0.05). Conclusion Taken together, these findings suggest that IL-36β could induce cell cycle arrest at S phase, inhibit keratin 10 and involucrin expressions and promote inflammatory activity in HaCaT cell lines.