Objective To investigate the role of peptidyl prolyl cis-trans isomerase 1 (Pin1) in mediating stemness of tumor cells and the molecular mechanism of inducing epithelial-mesenchymal transition (EMT) in cervical cancer cells. Methods The Siha and Hele cells of Pin1 low-expression stable transfection uterine cervical neoplasm cell lines were constructed using lentivirus transfection technology and were divided into control group (shPin1-NON), knockdown group 1 (shPin1-1) and knockdown group 2 (shPin1-2). Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expressions of Sex-determining region Y transcription factor 2 (SOX2), Aldehyde dehydrogenase 1A1 (ALDH1A1), and Cell adhesion molecule 44 (CD44). The serum-free spheroidization method was used to induce cervical cancer spheroids, with the adherent culture of cervical cancer cells as a control. Subsequently, Western blotting and qRT-PCR were employed to detect the expression of SOX2, ALDH1A1 and CD44 in both spheroid cells and adherent cells. Spheroid formation assay was used to detect the spheroid formation of cervical cancer cells after Pin1 knockdown. Transwell assay was used to detect the migration and invasion abilities of cervical cancer cells following down-regulation of Pin1. Western blotting and qRT-PCR were used to detect the expression of E-cadherin and N-cadherin attribute proteins in cervical cancer cells after transfection with pin1 low expression lentivirus. Western blotting and qRT-PCR were also used to assess the effects of Pin1 low expression on the expression levels of key proteins (c-Jun and c-Fos) of the transcriptional complex of Activator protein 1 (AP-1). Immunofluorescence combined with co-immunoprecipitation assays were conducted to detect the interaction and colocalization of Pin1 with c-Jun. Results In Siha and Hele cells, the mRNA and protein expression levels of Pin1, SOX2, ALDH1A1 and CD44 in shPin1-NON group were significantly higher than those in shPin1-1 group and shPin1-2 group (P<0.05). The expression levels of SOX2, ALDH1A1 and CD44 mRNA and protein in cervical cancer spheroid group were significantly higher than those in adherent cervical cancer cells (P<0.05). Compared with shPin1-NON group, the spheroidism and migration invasion abilities of shPin1-1 group and shPin1-2 group were significantly reduced (P<0.05). Compared with shPin1-NON group, the mRNA and protein expressions of E-cadherin in shPin1-1 and shPin1-2 groups were significantly increased (P<0.05), while the mRNA and protein expression levels of N-cadherin were significantly decreased (P<0.05). The mRNA and protein expression levels of c-Jun and c-Fos in shPin1-NON group were significantly higher than those in shPin1-1 group and shPin1-2 group (P<0.05). Conclusions Down-regulation of Pin1 can inhibit the stemness and migration invasion of cervical cancer cells, and Pin1 may mediate AP-1 to regulate the occurrence of stemness-induced epithelial-mesenchymal transition in cervical cancer cells.