Background: Chemoradiation is the standard neoadjuvant treatment for locally advanced rectal cancer. Microsatellite stable (MSS)/ mismatch repair proficient (pMMR) rectal cancer rarely responds to immune checkpoint inhibitor monotherapy, but combination with chemoradiation may improve sensitivity of MSS/pMMR tumors by inducing immune-stimulatory effects. Therefore, we aimed to investigate the efficacy and safety of patients treated with neoadjuvant preoperative short-course radiation followed by envafolimab plus CAPEOX for MSS/pMMR locally advanced rectal cancer. Methods: This is an open-label, single-center, phase II study. Patients (pts) with locally advanced rectal adenocarcinoma with MSS/pMMR were eligible. MMR protein expression was tested by immunohistochemistry (IHC), and MSI status was confirmed by NGS (next generation sequencing). All pts included in this study underwent neoadjuvant short-course radiation (total dose of 5×5Gy) in the first week after enrollment, then followed by 6 cycles of envafolimab (anti-PD-L1 inhibitor,150mg, d1, subcutaneous injection, QW) plus 2 cycles of CAPEOX in the next six weeks, and subsequently underwent total mesorectal excision (TME) in the ninth week. The primary endpoint is pathological complete response rate. Secondary endpoints include tumor regression grade (TRG), 3 year disease free survival, overall survival, toxicity, and quality of life (QoL). Results: This study intends to recruit 32 pts, and a total of 21 pts were enrolled from January 2022 to September 2022. Overall median age was 67 (42-79) years. 12 pts completed study designed treatment protocol (envafolimab plus CAPOX) followed with TME procedures, and 9 pts are still undergoing neoadjuvant treatment. All 12 pts achieved major partial response (MPR) evaluated by diffusion-weighted magnetic resonance imaging (DW-MRI), and 8 pts (8/12,76.6%) achieved pCR. During neoadjuvant treatment period, 20 pts (20/21, 95.2%) presented with treatment-related AEs (TRAEs) of any grade, 18 pts (18/21, 85.7%) had grade 1-2 and one each pts had grade 3 and 4 thrombocytopenia (2/21, 9.5%). The most common TRAEs were sensation of rectal tenesmus. Conclusions: Neoadjuvant preoperative short-course radiation followed by envafolimab plus CAPEOX in MSS/pMMR locally advanced rectal cancer achieved a promising pathologic response. Meanwhile, this combination neoadjuvant therapy is safe and worthy of application to clinical practice. (Funded by Sir Run Run Shaw Hospital Zhejiang University School of Medicine.) Clinical trial information: NCT05216653.