Aim:Mycobacterium tuberculosis in vitro biofilm is associated with the virulence and persistence capability. Our aim is to delineate factors involved in biofilms development. Materials & methods: We performed transposon mutants screen and found that mutation of MSMEG_3641, a homolog of M. tuberculosis Rv1836c, can change M. smegmatis colony morphology and biofilm. Results: MSMEG_3641 contains a vWA domain that is highly conserved among Mycobacteria. The phenotypes of MSMEG_3641 mutants include disrupted biofilm, weakened migration ability and changed colony morphology. All phenotypes might be contributed to the enhanced cell wall permeability and declined cell aggregation ability. Conclusion: To our knowledge, this is the first report concerning the mycobacteria Von Willebrand factor domain function, especially in colony morphology and biofilm development.