High mobility group box 1 (HMGB1) is a 25-kDa chromatin-associated protein that aids in transcription and DNA repair by directly binding to DNA and altering its conformation. Additionally, HMGB1 can act as an extracellular ligand. When released from dying or stressed cells, HMGB1 binds to the RAGE receptor and activates the p42/44 MAP kinase (MAPK) cascade. HMGB1 is overexpressed in many types of cancer and frequently associated with tumor stage and metastasis. This has predominantly been attributed to an autocrine function that drives MAPK pathway activity. However, by using tumor cells with activating MAPK pathway mutations, we have identified a role for HMGB1 in promoting metastasis and tumor growth that is independent of this pathway. In the absence of HMGB1, these tumor cells show defective in vitro migration as well as reduced metastasis and tumor growth in vivo despite high p42/44 phosphorylation. We found that semaphorin 3A (SEMA3A), previously shown to act as a suppressor of angiogenesis and migration, was highly increased during expression in the absence of HMGB1. SEMA3A/HMGB1 double knockdown rescued the migration defect in HMGB1 single knockdown cells. HMGB1 bound at the semaphorin 3A genomic locus, promoted hetrochromatin formation, and decreased occupancy of acetylated histones. Based on human tumor gene expression databases, HMGB1 was significantly inversely correlated with SEMA3A, suggesting that this mechanism may be more widely relevant in different cancer types.