Objectives: Osteosarcoma is a rare but aggressive bone tumor with limited therapeutic options and poor survival outcomes. The molecular mechanisms driving metastasis remain unclear. This study investigates regulators of anoikis resistance, a critical step in osteosarcoma metastasis. Methods: Osteosarcoma cells were cultured under attached or suspended conditions, followed by anoikis assays, RNA-seq, and immunoblot analyses. Candidate regulators were knocked down or overexpressed and tested for effects on anoikis. Their roles in metastasis were evaluated using a mouse model. Clinical relevance was assessed by IHC on patient tissues, survival analysis, and public datasets. Results: RNA-seq revealed upregulation of ECM-related pathways under suspension. Suspended osteosarcoma cells formed aggregates and acquired anoikis resistance. FN, TGFBR1, and NOX4 promoted aggregation and resistance; their knockdown impaired both, while exogenous FN restored resistance. TGFBR1 inhibition reduced FN expression and induced anoikis, rescued by TGFBR1 overexpression. NOX4 inhibition downregulated FN and TGFBR1, whereas NOX4 overexpression upregulated both and enhanced resistance. In vivo, knockdown of FN, TGFBR1, or NOX4 reduced lung metastases. Patient tissues showed positive correlations among NOX4, TGFBR1, and FN, and higher expression correlated with poor survival. Conclusions: The NOX4–TGFBR1–FN axis promotes anoikis resistance and metastasis in osteosarcoma. Targeting this pathway induces anoikis and suppresses metastasis, highlighting its potential as a therapeutic target.