Objective To determine the expression of anoikis factor Bcl2 inhibitor of transcription 1 (Bit1), epithelial-mesenchymal transformation (EMT) marker E-cadherin and P16^INK4a in tumor budding and central tumor of cervical squamous cell carcinoma, and to explore the significance of Bit1 and E-cadherin expression in the process of obtaining high invasiveness of cervical cancer and their relationship with P16^INK4a expression. Methods A total of 77 paraffin-embedded specimens of cervical squamous cell carcinoma were collected from the Department of Pathology of Gansu Provincial Cancer Hospital between 2014 and 2018. The expression levels of Bit1, E-cadherin and P16^INK4a in tumor budding and central tumor of these specimens were detected by immunohistochemistry. Taking the median scores of protein expression in the central tumor and tumor budding as dividing points, the specimens were divided into high expression group and low expression group. The differences of Bit1 and E-cadherin expression under different p16INK4a expression and their relationship with the clinicopathological characteristics of the patients were analyzed. The correlation between Bit1 and E-cadherin expression in central tumor and tumor budding was explored. The χ² test, continuous correction χ² test and Spearman rank correlation analysis were used for statistical analysis. Results In 77 cases of paraffin-embedded specimens of cervical squamous cell carcinoma, the high expression rates of P16^INK4a, E-cadherin and Bit1 in central tumor and tumor budding were 32.5% (25/77), 67.5% (52/77) and 63.6% (49/77), and 67.5% (52/77), 33.8% (26/77) and 37.7% (29/77), respectively, and the differences were significant (χ²=18.935, 17.561 and 10.391, all P<0.01). Both in central tumor and in tumor budding, there were no significant differences in Bit1 or E-cadherin expression between high and low P16^INK4a expression regions (all P>0.05). In central tumor, the low expression of Bit-1 was related to lymphovascular invasion and lymph node metastasis (χ²=5.053 and 4.400, both P<0.05). In tumor budding, the low expression levels of E-cadherin and Bit-1 were both associated with lymph node metastasis (χ²=5.580 and 7.573, both P<0.05). Spearman rank correlation analysis showed that there was positive correlation between E-cadherin and Bit1 expression in central tumor and tumor budding (r=0.287, P=0.011; r=0.236, P=0.039). Conclusion The increased invasiveness of cervical cancer may be related to the decreased expression of Bit1 and E-cadherin and the increased expression of P16^INK4a. Cervical cancer cells may acquire high invasiveness by inhibiting Bit1 to obtain anoikis resistance and affecting the EMT, but P16^INK4a is not involved in this process.