Abstract: Objective To investigate the expressions of long non-coding RNA antisense non-coding RNA in the INK4 locus (lncRNA ANRIL) and microRNA-181b-5p (miR-181b-5p) in the serum of patients with colon cancer complicated with type 2 diabetes mellitus (T2DM) and their correlations with lymph node metastasis. Methods A total of 117 patients with colon cancer complicated with T2DM in the hospital from May 2019 to August 2022 were selected as colon cancer with T2DM group and divided into lymph node metastasis group (n=56) and no lymph node metastasis group (n=61) according to the lymph node metastasis status. Additionally, 117 healthy volunteers with physical examinations in the same period were selected as control group. Reverse transcription real-time quantitative PCR was used to detect the relative expression levels of serum lncRNA ANRIL and miR-181b-5p. Logistic regression analysis was conducted to explore the factors influencing lymph node metastasis in patients with colon cancer complicated with T2DM. Receiver operating characteristic (ROC) curve was plotted to analyze the diagnostic values of serum lncRNA ANRIL and miR-181b-5p levels for lymph node metastasis in patients with colon cancer complicated with T2DM. Pearson correlation analysis was used to investigate the correlation between serum lncRNA ANRIL and miR-181b-5p expression in patients with colon cancer complicated with T2DM. Results Compared with the control group, patients in the colon cancer with T2DM group had significant increased serum lncRNA ANRIL level and decreased miR-181b-5p level (P<0.01). Compared with the no lymph node metastasis group, patients in the lymph node metastasis group had significant increased serum lncRNA ANRIL level and decreased miR-181b-5p level (P<0.01). There were significant differences in TNM stage, degree of differentiation, and glycated hemoglobin (HbA1C) between the lymph node metastasis group and the no lymph node metastasis group (P<0.05). History of diabetes, TNM stage, degree of differentiation, HbA1C, and lncRNA ANRIL were influencing factors for lymph node metastasis (P<0.05), while miR-181b-5p was a protective factor against lymph node metastasis (P<0.05). There was a negative correlation between serum lncRNA ANRIL and miR-181b-5p levels (r=-0.440, P<0.001). The ROC curve result showed that the areas under the curve (AUCs) for the diagnosis of lymph node metastasis by serum lncRNA ANRIL, miR-181b-5p, and their combination were 0.800, 0.837 and 0.930 respectively. The combined diagnostic value was significantly higher than that of lncRNA ANRIL (Z=2.956, P=0.003) and miR-181b-5p (Z=2.117, P=0.034) alone. Conclusion Patients with colon cancer complicated with T2DM have increased serum lncRNA ANRIL level and decreased miR-181b-5p level, and both two indexes are correlated with lymph node metastasis.