Pseudomonas aeruginosa (P. aeruginosa) is a common human opportunistic pathogen, and the biofilm formation could enhance its antibiotic tolerance. Chlorogenic acid (CA) has been reported to repress the biofilm formation of P. aeruginosa, so the present study aims to further explore the regulatory mechanism of CA, especially the effect of CA on the expression of Gac-Rsm (global secondary metabolism regulation system). The results showed that CA could repress the biofilm formation of P. aeruginosa, reduce exopolysaccharide production, although the transcription level of pslA (synthetase gene of key exopolysaccharide psl) was not significantly changed. The expression of RsmA (core regulator in Gac-Rsm system) was enhanced and the level of key signal molecule cyclic dimeric guanosine monophosphate (c-di-GMP) was reduced. It is suggested that CA represses the biofilm formation of P. aeruginosa via increasing the expression of RsmA.