The Gram-negative bacterium Acidovorax avenae subsp. avenae is the causal agent of bacterial brown stripe (BBS), which can cause severe diseases in many plants, including rice, with huge economic importance. Type IV pili (TFP) are hair-like appendages involved in several bacterial activities such as bacterial surface motility, surface adherence, colonization, biofilm formation, and virulence. The aim of our study is to characterize the association of A. avenae subsp. avenae TFP with BBS in rice. We generated a transposon (Tn5) mutant library. Then, an insertional mutagenesis on the background of this bacterium was identified as reduced pathogenicity. The confirmed inserted genetic region was into gene pilP, which encodes a TFP assembly protein. The pilP-deficient mutant strain seriously affected the motility twitching ability, biofilm formation and virulence. Collectively, our results clearly indicated that the pilP gene and TFP in A. avenae subsp. avenae play a key role in plant pathogenicity, twitching motility, and biofilm formation.