Previously, we have reported that 3-hydroxypropionate (3-HP) tolerance in Escherichia coli W is improved by deletion of yieP, a less-studied transcription factor. Here, through systems analyses along with physiological and functional studies, we suggest that the yieP deletion improves 3-HP tolerance by upregulation of yohJK, encoding putative 3-HP transporter(s). The tolerance improvement by yieP deletion was highly specific to 3-HP, among various C2-C4 organic acids. Mapping of YieP binding sites (ChIP-exo) coupled with transcriptomic profiling (RNA-seq) advocated seven potential genes/operons for further functional analysis. Among them, the yohJK operon, encoding for novel transmembrane proteins, was the most responsible for the improved 3-HP tolerance; deletion of yohJK reduced 3-HP tolerance regardless of yieP deletion, and their subsequent complementation fully restored the tolerance in both the wild-type and yieP deletion mutant. When determined by 3-HP-responsive biosensor, a drastic reduction of intracellular 3-HP was observed upon yieP deletion or yohJK overexpression, suggesting that yohJK encodes for novel 3-HP exporter(s).