Objective: To explore the mechanism of action of miR-29a in regulating COL6A6 (collagen type VI alpha 6 chain) and its correlation with the occurrence, development and prognosis of pituitary adenoma (PA). Methods: A total of 25 patients with non-invasive pituitary adenoma (NIPA) and 25 patients with invasive pituitary adenoma (IPA) who were treated in the Department of Rhinocranial Base Surgery, The First Affiliated Hospital of Kunming Medical University from March 2022 to March 2023 were consecutively selected. RT-qPCR (reverse transcription-quantitative polymerase chain reaction) and western blot were used to detect the expression levels of miR-29a and COL6A6 in pituitary adenoma tissues of the two groups, as well as in normal pituitary cells (MPC) and pituitary adenoma cells (AtT-20, HP75). miR-29a mimic, miR-29a inhibitor, control miRNA mimic and control miRNA inhibitor were transfected into PA cell lines, and RT-qPCR was used to detect the expressions of miR-29a and COL6A6. Dual-luciferase reporter gene assay was performed to verify the binding site relationship between miR-29a and COL6A6. CCK-8 (cell counting kit-8) assay was used to detect cell proliferation, and Transwell invasion assay was used to detect cell invasion ability. SPSS 27.0 was used for statistical analysis of the correlation between the differential expression of miR-29a and IPA, as well as the prognostic value of clinical diagnosis. Results: Compared with NIPA tissues, miR-29a was highly expressed in IPA tissues, with a statistically significant difference (p<0.001). Compared with normal pituitary cells (MPC), miR-29a was highly expressed in pituitary adenoma cells (AtT-20, HP75), and the difference was statistically significant (p<0.01). There was a negatively correlated targeted binding relationship between miR-29a and COL6A6. Overexpression of miR-29a promoted the proliferation and invasion of pituitary adenoma cells. The expression of miR-29a was associated with tumor size (χ2=4.056, p=0.021), Knosp grade (χ2=25.873, p<0.001) and total tumor resection rate (χ2=4.903, p=0.027). ROC (receiver operating characteristic) curve analysis showed that the expression level of miR-29a had certain value in evaluating the poor prognosis of PA (AUC=0.779, 95%CI: 0.638-0.920). Kaplan-Meier survival curve showed that the recurrence rates of the miR-29a low-expression group and high-expression group were (13.0% vs 51.9%), with a statistically significant difference (HR 3.882, 95%CI: 1.45-10.39, P=0.0069). Multivariate unconditional logistic regression analysis showed that total tumor resection rate (OR=0.009, 95%CI: 0.001-0.184, P=0.002) and miR-29a expression level (OR=15.325, 95%CI: 1.147-204.716, P=0.039) were independent risk factors affecting the prognosis of patients. Conclusion:miR-29a promotes cell proliferation and invasion by targeting COL6A6, which may provide new molecular markers and therapeutic targets for the diagnosis and treatment of pituitary adenoma.