Inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn's disease (CD). Glucocorticoids (GCs) are the most effective treatment for moderate to severe active UC. However, one-third of patients are not sensitive to GCs (i.e., they are GC resistant). The mechanism of GC resistance in IBD is unknown, and it remains unclear how to predict resistance in IBD patients. This study aimed to explore the possible correlation between miRNA expression and variability in GC-resistant and GC-sensitive patients with ulcerative colitis. A comparative serum microRNA analysis in GC-resistant and GC-sensitive patients with ulcerative colitis was conducted by microarray. Differential microRNA expression was further validated in serum samples by quantitative real-time PCR. We found that downregulated microRNAs had a significant correlation with several signal transduction pathways (the PI3K-Akt and MAPK signaling pathways) and target genes (HSP90B1, MAPK13, MAPK9, PIK3AP1 and TLR4) related to GC resistance. Eight downregulated microRNAs were chosen for further validation in 76 serum samples. The results showed that miR-16-2-3p, miR-30e-3p, miR-32-5p, miR-642a-5p, miR-150-5p, and miR-224-5p were significantly downregulated in the GC-resistant group. Receiver operating characteristic analysis showed that the area under the curves (AUCs) for those microRNAs were 0.94, 0.93, 0.85, 0.87, 0.92, and 0.99, with specificities of 97.30%, 89.20%, 59.50%, 73.00%, 97.30%, and 97.30% and sensitivities of 74.40%, 84.60%, 97.40%, 92.30%, 66.70%, and 89.70%, respectively. Our study provides preliminary evidence for the pathogenic mechanism of GC resistance and shows that serum microRNAs might serve as biomarkers for GC resistance in IBD.