Fish swim bladder-derived biomaterials are prospective cardiovascular materials due to anti-calcification, adequate mechanical properties, and good biocompatibility. However, their immunogenic safety profile, which primarily determines their feasibility as medical devices in clinical practice, remains unknown. Herein, the immunogenicity of glutaraldehyde crosslinked fish swim bladder (Bladder-GA) and un-crosslinked swim bladder (Bladder-UN) samples was examined using in vitro and in vivo assays according to ISO 10993-20. The in vitro splenocyte proliferation assay showed that cell growth was lower in the extract medium of Bladder-UN and Bladder-GA, compared to the LPS-or Con A-treated group. Similar results were obtained in in vivo assays. In the subcutaneous implantation model, the thymus coefficient, spleen coefficient and ratio of immune cell subtypes showed no significant difference between the bladder groups and the sham group. In terms of the humoral immune response, the total IgM concentration was lower in the Bladder-GA and Bladder-UN groups (988 ± 238 μg ml⁻¹ and 1095 ± 296 μg ml⁻¹, respectively) than that in the sham group (1329 ± 132 μg ml⁻¹) at 7 days. The total IgG concentrations were 422 ± 78 μg ml⁻¹ in Bladder-GA and 469 ± 172 μg ml⁻¹ in Bladder-UN at 30 days, which were slightly higher than that in the sham group (276 ± 95 μg ml⁻¹) but there was no significant difference compared with Bovine-GA (468 ± 172 μg ml⁻¹), indicating that these materials did not elicit a strong humoral immune response. Systemic immune response-related cytokines and C-reactive protein were stable during implantation, while IL-4 levels increased with time. The classical foreign body response was not observed around all the implants, and the ratio of CD163+/iNOS macrophages in Bladder-GA and Bladder-UN was higher than that in the Bovine-GA group at the implanted site at 7 and 30 days. Finally, no organ toxicity was observed in any of the groups. Collectively, the swim bladder-derived material did not elicit significant aberrant immune responses in vivo, giving strong confidence for its application in tissue engineering or medical devices. Furthermore, more dedicated research on immunogenic safety assessment in large animal models is encouraged to facilitate the clinical practice of swim bladder-derived materials.