Non-muscle myosin II (NMII) is a conserved force-producing cytoskeletal enzyme with important but poorly understood roles in cell migration. To investigate myosin heavy chain (MHC) phosphorylation roles in 3D migration, we expressed GFP-tagged NMIIA wild-type or mutant constructs in cells depleted of endogenous NMIIA protein. We find that individual mutation or double mutation of Ser-1916 or Ser-1943 to alanine potently blocks recruitment of GFP-NM-IIA filaments to leading edge protrusions in 2D, and this in turn blocks maturation of anterior focal adhesions. When placed in 3D collagen gels, cells expressing wild-type GFP MHC-IIA behave like parental cells, displaying robust and active formation and retraction of protrusions. However, cells depleted of NMIIA or cells expressing the mutant GFP MHC-IIA display severe defects in invasion and in stabilizing protrusions in 3D. These studies reveal an NMIIA-specific role in 3D invasion that requires competence for NMIIA phosphorylation at Ser-1916 and Ser-1943. In sum, these results demonstrate a critical and previously unrecognized role for NMIIA phosphorylation in 3D invasion.