Background: Anti-PD-1 mAb treatment has been shown to be effective in the treatment of esophageal cancer. Anlotinib has been approved in China as a second-line treatment for advanced esophageal cancer because of its good tolerance and efficacy. In the treatment of esophageal cancer, antiangiogenic target therapy combined with immunotherapy has been preliminarily recognized internationally. The purpose of this study is to investigate the efficacy and safety of anlotinib in combination with PD-1 inhibitors in second-line or later therapy during the treatment of esophageal cancer in the real world setting.(A LOT-EC3, NCT04966611). Methods: This is a prospective, multicenter real-world study. The esophageal cancer patients who received anlotinib in combination with PD-1 inhibitors in second-line or later therapy were enrolled. The primary endpoint was PFS (progression-free survival), and secondary endpoints were ORR (objective response rate), DCR (disease control rate) and OS (overall survival). The response to treatment was evaluated according to RECIST version 1.1. In addition, adverse events were evaluated by CTCAE v5.0. Results: From Jul 2020 to Sep 2021, 40 patients with a median age of 65.5 (range: 48̃81) were enrolled, in which the metastasis of more than 1 organ occurred in 62.5% of the 40 patients; the metastasis of more than 2 organs occurred in 37.5% (15 patients). 26 (65.0%) and 14 (35.0%) patients received anlotinib combined with PD-1 inhibitors as second line and third & above line therapy, respectively. Among all patients, 1 patient achieved complete response (CR), 11 patients partial response (PR), 23 patients stable disease (SD), illustrating an ORR of 30.0% and a DCR of 87.5%. In the second line therapy, ORR was 30.8 %, DCR was 92.3%. In the third & above line, ORR with 28.6%, DCR with 78.6% was observed. Median PFS was not reached. For all the 40 patients, 27 of which were treated with Camrelizumab, 10 were treated with Sintilimab, other 3 patients were treated with other kinds of PD-1 inhibitors. There was no significant difference in DCR between different PD-1 inhibitors. Treatment related adverse events (trAEs) occurred in 22 (55%) patients. 4 patients had grade 3 or more trAEs. No patient died due to trAE. Conclusions: Anlotinib combined with PD-1 inhibitors is found to be a reasonable option in second-line or later therapy for the treatment of advanced esophageal cancer. To our best knowledge, this is the first summarized real-world study on anlotinib combined with PD-1 inhibitors in advanced esophageal cancer. The treatment effects are similar to the outcomes in comparable clinical trials, which support the further recommendation of anlotinib combined with PD-1 inhibitors in the treatment of esophageal cancer. Clinical trial information: NCT04966611.