Background: Combining CAR-T with programmed cell death-1 (PD-1) inhibition may be a more efficacious approach for treatment of solid tumors. This clinical study was designed to assess the safety and tolerability of PD-1 knockout MUC1-Targeted CAR-T engineered Cells to treat advanced esophageal cancer. Methods: Patients with advanced esophageal cancer were recruited according to the criteria in NCT03706326. PD-1 gene knockout (KO) MUC1-targeted CAR-T cells were prepared by constructing MUC1-specific CARs using the SM3 scFv in patient-derived T-cells, and PD-1 gene KO was achieved in these CAR positive T cells using the CRISPR-Cas9 system. Efficiency and validation were confirmed by sequencing and flow cytometry. MUC1-CAR+/PD-1- KO engineered T cells at a starting dose of 5x10⁷ were infused over 60 min. The dose was escalated to 5x10⁹ cells. Following treatment, the safety and tolerability of dose of the therapeutic cells were assessed using CTCAE v4.0. Patients’ general condition, levels of lymphocytes, IL-6, hs-CRP, PCT, CYFRA21, NSE(E), and SCC were monitored at regular intervals. Circulating CART cells were checked regularly. Tumor size were examined by MRI/CT scans. Efficacy was also assessed. Results: A total of 9 patients (aged 48 to 80) diagnosed with esophageal cancer/esophageal squamous cell carcinoma (IIIb to IV), were included in this study. All participants received at least one cycle of CART cell treatment. Among the 9 treated patients, 4 received 1 cycle, other 5 received 2, 3, 4, 7 and 10 cycles, respectively. The most common adverse events (AE) were acute fever (8 patients, 38.0-40.5 C◦), chills (5 patients, occurred in the subsequent cycle of infusion), skin rash (4 patient). No grade 3-5 AEs were found, and no cytokine release syndrome (CRS) was observed. Of the 9 assessed patients, 6 had stable disease (SD) while 3 had progressive disease. All patients had significant symptom improvements after infusion. Circulating CART cells gradually declined after infusion; the number dropped down to approximately 25% in 4 months after one cycle treatment indicating the necessity of receiving more cycles. Patients who received multiple cycles of infusion showed long-term and steady SD status. Two patients who received a multiple cycle of CAR-T cells had an overall survival (OS) of 24 months. Conclusions: Our data suggests that the treatment of patients with advanced esophageal cancer with PD-1 disrupted MUC1-targeted CAR-T cells is safe and well-tolerated by all patients. No CRS and other serious EAs were observed throughout the study. The efficacy of this unique combined therapy is currently being assessed. Our limited data indicate that the OS of the patients extended significantly in the treated patients. Two out of 6 patients with an OS of greater than 24 months, an outstanding clinical outcome. Clinical trial information: NCT03706326.