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Simple Summary: With a mass production scale and important economic and nutritive value, red sea bream (Pagrosomus major) is one of the most popular farmed marine teleost fish species. However, outbreaks of diseases have produced huge losses to the aquaculture industry in recent years. Fish cell lines are suitable models to study fish physiology, virology, immunology and toxicology. As the main target organ for Nervous necrosis virus infection, primary cell cultures of brain tissue were initiated for the first time from Pagrosomus major in this study. Our data showed that this cell line could provide wide applications for virology and toxicology for Pagrosomus major. RSBB cells are susceptible to Nervous necrosis virus, Singapore grouper iridovirus, Infectious spleen and kidney necrosis virus and Siniperca chuatsi rhabdoviruses infection. Moreover, single exposure of RSBB cells to metal Cd resulted in cytotoxicity and could induce cell apoptosis and necrosis. This study established and characterized a new marine fish brain cell line and demonstrated its suitability as a model for fish virology and toxicology. Red sea bream (Pagrosomus major) is one of the most popular farmed marine teleost fish species. Fish cell lines are becoming important research tool in the aquaculture field, and they are suitable models to study fish virology, immunology and toxicology. To obtain a Pagrosomus major cell line for biological studies, a continuous cell line from brain of red sea bream (designated as RSBB cell line) was established and has been successfully subcultured over 100 passages. The RSBB cell line predominantly consisted of fibroblast-like cells and multiplied well in M199 medium supplemented with 10% fetal bovine serum at 28 °C. Karyotyping analysis indicated that the modal chromosome numbers of RSBB cells was 48. After transfection with pEGFP-N1, RSBB cells showed bright green fluorescence with a transfection efficiency approaching 8%. For toxicology study, it was demonstrated that metal Cd could induce cytotoxic effects of RSBB cells, accompanied with a dose-dependent MTT conversion capacity. Morphologically, cells treated with metal Cd produced rounding, shrinking and detaching and induced both cell apoptosis and necrosis. For virology study, the RSBB cells were highly susceptible to Nervous necrosis virus (NNV) and Singapore grouper iridovirus (SGIV) with steady titers (i.e., 108.0~8.3 TCID50 mL−1 and 107.0~7.2 TCID50 mL−1 respectively). Furthermore, an obvious cytopathic effect (CPE) could be observed in RSBB cells infected with Infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdoviruses (SCRV). Meanwhile, all the infections were confirmed by polymerase chain reaction. The new brain cell line developed and characterized from red sea bream in this study could be used as an in vitro model for fish studies in the fields of toxicology and virology. [ABSTRACT FROM AUTHOR]