Background: Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal cancer in China. Its prognosis remains poor and neoadjuvant treatment may improve the prognosis for resectable ESCC. The combination of immunotherapy with cytotoxic agents has shown encouraging antitumor activity in multiple tumors but data are lacking to support this approach as a neoadjuvant strategy for locally advanced ESCC. We conducted a prospective clinical trial of pre-operative pembrolizumab with chemotherapy in patients with locally advanced ESCC. Methods: Patients with clinical stage IIB-IVA ESCC eligible for resectable surgery were prospectively enrolled. Patients received neoadjuvant pembrolizumab (200mg) and chemotherapy (docetaxel 75mg/m² plus cisplatin 75mg/m² or carboplatin area under curve 4-5) every 3 weeks for four cycles before surgery. The primary endpoint was pathologic complete response (pCR) rate and radical (R0) resection rate. Secondary endpoints include Disease Free Survival (DFS), adverse events, and the correlation between biomarkers and immunotherapy efficacy. Results: From August 2020 to January 2022, 27 eligible patients participated the study. Twenty-four patients have completed four cycles of neoadjuvant therapy, among whom 22 patients were radiologically evaluable after neoadjuvant therapy. The objective response rate (ORR) was 68.2% and disease control rate was 100%. Among 22 patients with resectable tumors, 14 patients underwent McKeown MIE and 5 patients are still waiting for the surgery. Three other patients who achieved partial response (PR) refused surgery due to fear of complications. For 14 patients who underwent esophagectomy within 29-74 (median44) days after neoadjuvant treatment, all achieved radical resection. pCR (ypT0N0) was observed in 4 patients (4/14, 28.6%). Three patients (3/14,21.4%) had a College of American Pathologists (CAP) score of 1, one (1/14, 7.1%) had a CAP of 2, and 6 (6/14, 42.9%) had a CAP of 3. Most treatment-related AEs (TRAEs) were grade 1 or 2. Grade 3/4 TRAEs of leucopenia, decreased neutrophil count and dermatitis occurred in 9 (33.3%), 14 (51.9%), and 1 (3.7%) cases, respectively. No surgical complication was observed. Conclusions: Neoadjuvant pembrolizumab plus chemotherapy has achieved satisfactory initial efficacy and safety results in locally advanced ESCC. A phase III randomized controlled trial is required to confirm the clinical benefits of this neoadjuvant therapy.