The cytoplasmic distribution of cellular structures is known to depend on the balance between plus- and minus-end-directed motor complexes. Among the plus-end-directed kinesins, kinesin-1 and -2 have been implicated in the outward movement of many organelles. To test for a role of kinesin-1 previous studies mostly relied on the overexpression of dominant-negative kinesin-1 constructs. The latter are often cytotoxic, modify the microtubule network and indirect effects related to altered microtubule dynamics should be excluded. In the present study we present a novel kinesin-1 construct, encompassing the first 330 amino acids of kinesin heavy chain fused to GFP (kin330-GFP) that does not alter microtubules upon its overexpression. Kin330-GFP functionally inhibits kinesin-1 because it induces the peri-nuclear accumulation of mitochondria and intermediate filaments. Using this construct and previously established siRNA-mediated knock-down of kinesin-2 function, we assess the role of both motors in the subcellular distribution of distinct steps of the vaccinia virus (VV) life cycle. We show that kinesin-1, but not kinesin-2, contributes to the specific cytoplasmic distribution of three of the four steps of VV morphogenesis tested. These results are discussed with respect to the possible regulation of kinesin-1 during VV infection.