Pseudomonas aeruginosa coordinates the secretion of virulence factors through quorum sensing. Quorum sensing inhibitors can attenuate the pathogenesis of bacteria and help the immune system to eradicate them without targeting the bacterial growth. This study aimed to explore the anti-quorum sensing and anti-virulence activities of sitagliptin against P. aeruginosa PAO1 strain. Sub-inhibitory concentration of sitagliptin significantly inhibited the virulence factors pyocyanin, hemolysin, protease and elastase in addition to blocking swimming, swarming and twitching motilities and biofilm formation. In silico analysis showed that sitagliptin interacted with LasR receptors by hydrogen bonding and hydrophobic interaction, mainly with the amino acids leucine present at positions 40 and 125, tyrosine at position 56, serine at position 129, tryptophan at position 60, alanine at position 50 and phenyl alanine at position 101. qRT-PCR confirmed the anti-quorum sensing activity by reducing the expression level of QS genes lasI, lasR, rhlI, rhlR, pqsA and pqsR. In conclusion, sitagliptin is a novel anti-quorum sensing agent that can be used for treating P. aeruginosa infections.