Tisochrysis lutea is a highly nutritious marine microalga that has various applications in aquaculture and biotechnology. However, the effects of T. lutea extract (TLE) on osteoarthritis (OA) pathogenesis remain unexplored. In this study, we aimed to determine the effects of TLE on OA development. We found that TLE inhibits the expression of matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) activity in an OA mouse model generated by the destabilization of the medial meniscus (DMM) surgery. In vivo assays of the OA model mice demonstrated that TLE has a protective effect against cartilage destruction by inhibiting MMP3 and MMP13 expression. To enable the medical use of TLE, the components of TLE were characterized using high-performance liquid chromatography (HPLC) analysis. Interestingly, we found that Fucoxanthin accounts for 41.2% of TLE and showed anti-catabolic and antioxidant effects under IL-1β-treated in vitro conditions. RNA sequencing analysis showed that fucoxanthin decreased p38, NF-κB, and JNK signaling pathway gene expression, all of which are activated by IL-1β. Furthermore, in vivo analysis showed that fucoxanthin inhibited the IL-1β-stimulated phosphorylation of p65, JNK, and p38. These results highlight new possibilities for the use of TLE as a source of fucoxanthin, an antioxidant, for OA treatment.