[Objective] To investigate the inhibitory activity of trans-cinnamaldehyde (TC) and cinnamaldehyde (CD) on spoilage and biofilm formation of Pseudomonas isolated from spoiled beef. [Methods] The minimal inhibitory concentration (MIC) on Pseudomonas lundensis was measured by plate counting. Effects of sub-MIC two cinnamaldehyde on the biofilm formation and extracellular enzymes were evaluated by crystal violet staining, bead vortexing, confocal laser scanning microscope, folin method. The effects of sub-MIC TC and CD on the gene expressions of lapA, fliC, aprX and lip were detected by real-time quantitative PCR. [Results] MIC of TC and CD was 200 μg/mL and 225 μg/mL, respectively. Cinnamaldehyde at sub-inhibitory concentration of 1/8 MIC, 1/4 MIC, 1/2 MIC decreased significantly the biofilm biomass and adhesion of P. lundensis. Compared to the control, the biofilm of P. lundensis decreased by 60.27% and 52.05%, when supplemented with exogenous 1/2 MIC of TC or CD, respectively. The 1/2 MIC of TC and CD decreased the bacterial adhesion by 56.35% and 61.10%, respectively. The treatment by TC or CD resulted in the thinner biofilm thickness of P. lundensi, even partial biofilm cell was killed by TC. Two cinnamaldehyde strongly inhibited swimming motility, and the inhibitory rates were 58.08% and 42.29% by 1/2 MIC of TC and CD, respectively. Furthermore, protease of P. lundensis decreased by 61.90% and 76.19%, while lipolytic activity inhibited by 40.17% and 47.01% in the presence of 1/2 MIC of TC and CD, respectively, indicating that sub-MIC TC or CD markedly repressed the two extracellular enzymes activity. Cinnamaldehyde caused greatly down-regulated gene expressions of lapA, fliC, aprX and lip (P<0.05), except no significant difference in lapA gene treated by 1/8 MIC of CD. The expressions of lapA, fliC, aprX and lip decreased to 0.05-0.16 and 0.02-0.12 fold in P. lundensis treated with 1/2 MIC of TC and CD (P<0.05), respectively. [Conclusion] Two cinnamaldehyde isomers at sub-MIC significantly repressed biofilm formation and spoilage of P. lundensis. TC had a stronger inhibitory effect on biofilm formation, while CD was more effective in decrease of extracellular enzymes, which was closely associated with down-regulation of expression of related genes by cinnamaldehyde.