The vulnerable status of the Amazon manatee, Trichechus inunguis, indicates the need to seek measures to guarantee its conservation. In this context, the cultivation of cells in vitro is a strategy that should at least guarantee the preservation of their genetic material. Thus, we established for the first time a primary culture of Amazonian manatee fibroblasts (TINsf) from a skin biopsy of a young male. Karyotypic analysis of the 3rd, 7th, and 12th passages confirmed the taxonomic identity of the species T. inunguis (2n = 56/NF = 92) and indicated that this culture presents genomic stability. Gene and protein expression of vimentin at the 13th passage show the predominant presence of fibroblasts in TINsf. To test the cell line’s responsiveness to materials and demonstrate a possible application of this culture, it was exposed to andiroba seed oil (ASO), and its viability and proliferative capacity were evaluated. ASO demonstrated toxic effects at the highest concentrations and longest exposure times tested, reproducing results observed in human cultures, indicating the applicability of TINsf in toxicological and biotechnological studies. After cryopreservation, the TINsf line maintained its proliferative potential, indicating the establishment of a new culture available for future studies.