As a rate-limiting step in metastasis, metastatic colonization requires survival signals from supportive stroma. However, the mechanisms driving this process are incompletely understood. Here, we showed that the proliferation of B16F10 cells was promoted when cocultured with lung fibroblasts. Meanwhile, co-injection of B16F10 tumor cells with mouse lung fibroblasts significantly increased lung metastasis. Based on GEO database, we identified stearoyl-CoA desaturase 1 (SCD1) as a novel factor promoting metastatic colonization. Importantly, we found that fibroblast-secreted cathepsin B (CTSB) induced the upregulation of SCD1 in B16F10 through Annexin A2 (ANXA2) and PI3K/Akt/mTOR pathway. The elevated SCD1 induced a higher ratio of monounsaturated fatty acids to saturated fatty acids in B16F10 cells. The changes in fatty acid composition contributed to tumor cell proliferation and metastatic colonization. Furthermore, targeting SCD1 effectively inhibited lung metastasis and prolonged the overall survival of mice. Meanwhile, the expression of SCD1 was negatively correlated with disease-free survival in five types of cancer patients. Collectively, our study identifies SCD1 as a critical modulator of tumor cell proliferation that is activated by cathepsin B, secreted by lung fibroblasts at the metastatic niche. Our novel findings provide potential therapeutic targets to prevent tumor metastasis.