Abstract: Influenza virus is an RNA virus that classified into 4 types, A, B, C, and D, where influenza A and B virus infection may cause human acute respiratory tract infection and nearly 0.3 million deaths annually. The life cycle of influenza virus infection is highly dependent on the host response, demonstrating an important strategy of developing anti-influenza agents that target the host factors. This research utilized a transcriptome signature reversion (TSR) strategy to discover a list of multi-host-factor-target anti-influenza agents and determined their anti-influenza activities in vitro. BIX02189 was discovered and exhibited broad spectrum anti-influenza activity, with half maximal effective concentration (EC₅₀) of 17.1 μmol·L^-1 against influenza A virus H1N1 (A/Puerto Rico/8/1934) and 9.4 μmol·L^-1 for influenza B virus (B/Jiangxi Xinjian/BV/39/2008). The anti-influenza A virus activity of BIX01289 is stronger than the positive control ribavirin with EC₅₀ of 97.9 μmol·L^-1 for influenza A virus H1N1 (A/Puerto Rico/8/1934). According to the unsupervised transcriptomic profile similarity clustering analysis, BIX02189 was considered to inhibit viral protein synthesis and release of influenza virus mainly through inhibiting the Raf/MEK/ERK cascade, revealing its potential mechanism of inhibiting influenza virus infection.