Acinetobacter baylyi ADP1 has been studied in laboratories because of their competence for natural transformation and ability to adapt to different environmental conditions. A previous study has found 30 different genes in A. baylyi ADP1 that are induced by starvation during the long term stationary phase. ACIAD0167 is one of them, encoding a Vgr-like protein. The goal of this study was to test whether ACIAD0167 and other genes in its operon (ACIAD0166, ACIAD0168 and ACIAD0169) are required for twitching motility, or for surviving stressful conditions including heat shock, desiccation and DNA damage. Our study found that ACIAD0167 and the other three genes in the operon play a role in twitching motility in A. baylyi but apparently not in other phenotypes. The likely first gene in the operon, ACIAD0166, was cloned into wild-type ADP1 and over-expression of the gene caused a smaller twitching zone than one produced by wild type cells, further implicating the role of ACIAD0167-containing operon in twitching motility. These results indicate that ACIAD0166, ACIAD0167, ACIAD0168, and ACIAD0169 genes encode proteins, and should no longer be considered “hypothetical” genes. We also found a novel link among ACIAD0167, twitching motility and the type VI secretion system (T6SS). ACIAD0167 is found in the STRING network to be associated with genes involved the T6SS, whose structure resembles an inverted bacteriophage tail.