Background <p>Myxoid stroma in the tumor stroma has been suggested to play a key role in cancer progression; however, its underlying mechanisms remain unclear. This study aimed to examine the relationship between myxoid stroma, identified in hematoxylin and eosin (HE)-stained tissue, and tumor-infiltrating lymphocytes (TILs), assessed through immunohistochemical staining. Automated image analysis was employed to evaluate these features, and their clinical significance in esophageal squamous cell carcinoma (ESCC) was investigated.</p> Methods <p>A total of 139 ESCC patients who underwent esophagectomy were included. From each patient, three serial sections from paraffin blocks were prepared and stained with CD3, HE, and CD8, respectively. An algorithm for identifying myxoid stroma was applied to HE slides, and the resulting data were mapped onto the corresponding CD3 and CD8 slides to analyze the spatial relationship between these pathological findings.</p> Results <p>The myxoid-high group, comprising 76 cases, had significantly deeper tumor depth compared to the myxoid-low group, which included 63 cases (<i>p</i> &lt; 0.05). CD3⁺ cell density was significantly lower in the myxoid-high group compared to the myxoid-low group (<i>p</i> &lt; 0.01). Within the myxoid stroma regions, CD3⁺ cell density was significantly lower than in regions outside the stroma (<i>p</i> &lt; 0.01). The mean distance of all CD3⁺ cells from the edges of the myxoid stroma was significantly greater than that from the tumor margin (<i>p</i> &lt; 0.01). Similar results were observed for CD8⁺ cells.</p> Conclusions <p>In conclusion, this study demonstrated that myxoid stroma in the invasive front of ESCC is closely associated with the density of CD3⁺ and CD8⁺ positive lymphocytes.</p>