Goals This multicenter, retrospective research aims to compare effectiveness and tolerability of paclitaxel-based chemotherapy combined with immune checkpoint inhibitors (ICIs) versus chemotherapy alone as a first-line treatment of HER2-negative AGC in a real-world setting. Background Platinum-based chemotherapy combined with ICIs is now becoming the standard first-line therapy of HER2-negative AGC. In China, paclitaxel has shown good efficacy and tolerability in AGC as an alternative for first-line therapy. Combining ICIs with paclitaxel-based chemotherapy may lead to improved tumor immune microenvironment, but evidence in paclitaxel combing with ICIs as first-line regimen is lacking. Methods 86 patients with HER2-negative AGC were enrolled from 2017 to 2022. Among them, 57 patients received paclitaxel-based chemotherapy plus ICIs, and 29 patients received paclitaxel-based chemotherapy alone. We compared the efficacy and incidence of adverse events between the two therapy options. Results Significant improvements in median PFS (8.77 versus 7.47 months; P=0.048) and median OS (15.70 versus 14.33 months; P=0.048) were observed in the ICIs combined with paclitaxel-based chemotherapy group. Meanwhile, the ICIs plus chemotherapy group demonstrated significantly improved ORR (50.9% vs. 27.6%; P=0.039) and DCR (98.3% vs. 82.8%; P=0.015), and the side effects were tolerable. Conclusion In summary, for HER2-negative AGC, ICIs plus paclitaxel-based chemotherapy is effective with mild toxicities, which should be considered as an alternative first-line therapy regimen.