Background: Programmed death-1 (PD-1) inhibitors alone have modest activity in the treatment of most STS. A possible synergistic effect of combined trabectedin with PD-1 inhibitor nivolumab has been previously reported. Herein we evaluated the efficacy and safety of trabectedin plus nivolumab as a second-line treatment of patients with anthracycline-pretreated metastatic or inoperable STS. Methods: The prospective, explorative, two-group, non-randomized phase II NiTraSarc trial enrolled patients with advanced lipo- or leiomyosarcomas (Group A; GA) or with non-L-sarcomas (Group B; GB). Patients were initially treated with 3 cycles of trabectedin 1.5 mg/m², followed by the combination of trabectedin 1.5 mg/m² plus nivolumab 240 mg in a so-called “late combination cohort” (LCC) for up to 16 cycles. After positive results of a preplanned interim analysis, patients received the combination therapy already starting from Cycle 2 in an “early combination cohort” (ECC). Primary efficacy endpoint was progression-free survival rate after 6 months (PFSR6) according to RECIST v.1.1. A central pathological assessment (CPA) was done from tumor specimen from all patients. Updated results according to cpa are presented. Results: A total of 92 patients were recruited in the trial: 43 patients in GA and 49 in GB. In GA, 28 patients (63%) had leiomyosarcoma and 15 (37%) had liposarcoma. Most common sarcoma types in GB were pleomorphic (n = 12), spindle cell (n = 11), fibromyxoid (n = 6), synovial (n = 5) and epithelial (n = 4) sarcoma. After median follow up of 16.6 months, overall PFSR6 as per CPA in GA was 47.6% (60% in LCC vs 36.4% in ECC) and 14.6% in GB. Median PFS was numerically higher in GA compared to GB (5.5 vs 2.3 months) and even longer in LCC vs ECC (9.8 vs 4.4 months). Median overall survival was more than three times longer in GA vs GB (18.7 vs 5.6 months) and, again, longer in LCC vs ECC (24.6 vs 13.9 months). Safety trabectedin and nivolumab was consistent with the safety profiles of each drug alone with no relevant new findings for the combination or between LCC and ECC. Conclusions: Our study confirms the activity of trabectedin plus nivolumab, particularly in patients with lipo- or leiomyosarcomas. There is a significant difference between ECC and LCC in terms of PFSR6, PFS and OS. The results in patients with non-L-sarcomas do not justify further investigation of this combination. Clinical trial information: NCT03590210.