Background: Neoadjuvant chemoradiation therapy followed by radical surgery has been extensively recommended for locally advanced esophageal squamous cell carcinoma (ESCC). However, toxicities and challenges to the subsequent surgery limit its wide clinical application. Immune checkpoint inhibitors combined with antiangiogenesis have synergistic effects and have shown good therapeutic effects in previous studies, as well as fewer side effects. Here, we tested a novel combination of anlotinib (an antiangiogenic kinase inhibitor) and penpulimab (an anti-PD-1 antibody), without the use of radio- or chemotherapy, for the neoadjuvant therapy of locally advanced ESCC. Methods: In this prospective, single-arm, phase II clinical trial (ChiCTR2200064848), patients(pts) with resectable locally advanced ESCC (clinical stage III-IVa) were recruited. The study was designed using a two-stage minimax approach. The estimated sample size was 40 pts would provide 80% power at a one-sided alpha error of 5%, threshold pathological complete response (pCR) of 16% and expected pCR of 30%. Here, the first-stage data was included. Notably, 85% (22/25) enrolled pts had medical contraindications (including cardiopulmonary diseases or other underlying medical conditions, along with poor nutritional status and so on). Pts received neoadjuvant therapy with anlotinib (8 mg, po, D1-14, Q3W) combined with penpulimab (200 mg, ivgtt, Q3W) for 2 cycles. Six to eight weeks later, pts underwent radical surgery after exclusion of contraindications. The primary endpoint was pathologic complete response (pCR) rate. Secondary endpoints included tumor regression grade (TRG), R0 resection rate, objective response rate (ORR) and safety profile. Results: From Jan 2023 to Sep 2023, 25 pts (19 males and 6 females) were enrolled with a median age of 67 years (range: 50-74). All pts were available for efficacy assessment and 16 were evaluable for the primary end point. The rest of the pts are waiting for surgery. As of 15 Sep 2023, 19 pts underwent esophagectomy and R0 resection rate was 87.5%. The pCR rate was 18.8% (3/16) and 31.3% of pts had a major pathologic response (MPR). The ORR and DCR were 48.0% and 96.0%, respectively. 84.0% of pts experienced grade 1-2 treatment-related adverse events (TRAEs), 3 (12.0%) pts experienced grade 3 TRAEs (2 elevated blood pressure and 1 elevation of liver enzymes). The safety profile revealed that the most common TRAEs (≥10%) included hypertension (28.0%), hand-foot syndrome (20%), fatigue (20%), abdominal pain (12%), toothache (12%). Conclusions: The preliminary results of this study show promising safety and efficacy of anlotinib combined with penpulimab as neoadjuvant for pts with ESCC, especially for pts not suitable for neoadjuvant chemoradiation or chemotherapy. More ESCC pts would be recruited in the future. Clinical trial information: ChiCTR2200064848.