Summary Movement proteins ( MP s) encoded by plant viruses interact with host proteins to facilitate or interfere with intra‐ and/or intercellular viral movement. Using yeast two‐hybrid and bimolecular fluorescence complementation assays, we herein present in vivo evidence for the interaction between Alfalfa mosaic virus ( AMV ) MP and Arabidopsis P atellin 3 ( atPATL3 ) and P atellin 6 ( atPATL6 ), two proteins containing a S ec14 domain. Proteins with S ec14 domains are implicated in membrane trafficking, cytoskeleton dynamics, lipid metabolism and lipid‐mediated regulatory functions. Interestingly, the overexpression of atPATL3 and/or atPATL6 interfered with the plasmodesmata targeting of AMV MP and correlated with reduced infection foci size. Consistently, the viral RNA levels increased in the single and double Arabidopsis knockout mutants for atPATL3 and atPATL6 . Our results indicate that, in general, MP – PATL interactions interfere with the correct subcellular targeting of MP , thus rendering the intracellular transport of viral MP ‐containing complexes less efficient and diminishing cell‐to‐cell movement.