Background: Neoadjuvant immunotherapy and chemotherapy have shown promise in improving outcomes for patients with locally advanced ESCC, but the response to treatment can vary among patients. Therefore, there is a need to identify predictive biomarkers that can guide treatment decisions and improve patient outcomes. The infiltration of immune cells within the tumor microenvironment has been shown to be an important factor in cancer progression and response to treatment. In this study, we sought to explore the immune microenvironment prospectively related to treatment response of Neoadjuvant immunotherapy by scRNAseq. Methods: This was a single-arm phase II clinical trial, planned to enroll 20 locally advanced ESCC patients (cT2-4N0-1M0). After neoadjuvant chemotherapy (paclitaxel-albumin + carboplatin) combined with tislelizumab, patients received minimal invasive esophagectomy (MIE).Prior to neoadjuvant therapy, fresh tumor tissues were obtained via endoscopy and used for scRNAseq analysis, to evaluate the predictive capacity of the immune microenvironment. Pathological outcomes were assessed and divided into pCR, MPR defined as <10% residual tumor excluding pCR, and non-MPR defined as >10%. Results: Out of 18 enrolled patients, 15 completed neoadjuvant therapy and MIE surgery since March 2022. Of these 15 patients, 3 achieved pCR, 2 got MPR, and 10 were non-MPR, with a total of 51,208 single cells sequenced. Both groups showed similarly low expression of PD-L1 (5.7% vs. 2.3%). Comparing pCR to non-MPR patients, the former showed a significant increase in T lymphocytes (CD8+(17.1% vs.1.1%), Regulatory T(20.2% vs.4.0%)), plasma cell(22.0% vs.8.3%), and mature dendritic cell(1.5% vs.0.5%); a significant decrease in B cells(0.9% vs. 1.9%), neutrophils(1.7% vs.11.0%), and tumor cell, as well as a similar expression of macrophage(5.6% vs.6.6%) and monocyte, indicating a clear difference in the immune cell infiltration landscape between two groups. Furthermore, we identified a new subtype of plasma cell which was highly enriched in the non-MPR group, even though the pCR group showed a great increase in plasma cells in total. Conclusions: This study demonstrates the heterogeneity of baseline immune cell infiltration landscape in Locally Advanced ESCC, in which pCR and non-MPR patients exhibited entirely different profiles, and provide a potential predictor of response to neoadjuvant immunotherapy. Our findings suggest that baseline immune cell infiltration status, rather than pd-L1 expression, could accurately predict pCR or non-MPR before treatment, which could guide personalizing and minimize the need for trial-and-error approaches.