Background: Squamous cell carcinoma of the anus (SCCA) is a rare malignancy with limited treatment options for metastatic patients. Immune checkpoint inhibitors (ICI) have been studied on few prospective trials, but its efficacy seems low. Methods: We retrospectively collected clinical, radiological, and laboratory data from patients (pts) diagnosed with metastatic SCCA who received nivolumab as 2^nd or 3^rd line therapy at the Oncology Unit of Bologna University Hospital from October 2017 to November 2023. We analyzed the correlation of clinical-pathological features with the overall response rate (ORR), overall survival (OS), and progression-free survival (PFS). Results: We enrolled 15 pts diagnosed with relapsed/metastatic SCCA; pts characteristics are summarized in Table 1. Nivolumab was administered as a 2^nd line treatment in 13 pts, while 2 pts received ICI in the 3^rd line. Overall, median OS was 8.3 months (mo.) (95% CI 4.37-NA) and median PFS was 4.37 mo. (95% CI 3.67-NA). Of note, ORR was 40%, compared with the 10% ORR seen in the few prospective phase II trials available. Pts with ECOG PS ≥ 2 showed a lower ORR and resulted in worse OS and PFS. ECOG PS 0 pts showed a median OS of 19.17 mo., PFS of 5.17 mo., and ORR of 75%. ECOG PS 1 pts had an OS of 10.73 mo., a PFS of 4.98 mo., and an ORR of 38%, while ECOG PS 2 pts showed 4.37 mo. of OS and PFS with no response seen. The number of metastatic sites at baseline has been shown to be associated with OS. Pts with ≥ 3 metastatic sites showed an OS of 5.17 vs 10.73 mo. for pts with < 3 sites. Overall, no complete response was seen; however, we identified 4 long-responders with a median treatment duration of 15.8 mo. (8.3 - 26.5). All long-responders were HPV-positive and had no liver metastases at the time of treatment initiation. PD-L1 evaluation and molecular characterization is ongoing. Nivolumab was well-tolerated with 5 pts experiencing immune-related adverse events: 4 pts showed G1 toxicities according to the CTCAE, version 5.0 (thrombocytopenia, hypothyroidism and rash) while 1 patient had G4 myopathy caused a treatment discontinuation. Conclusions: ICI is a possible treatment option with a favorable safety profile in pretreated SCCA although a better selection of patients is required.