The stress response of Escherichia coli to 3-hydroxypropanoic acid (3-HP) was elucidated through global transcriptomic analysis. Around 375 genes showed difference of more than 2-fold in 3-HP-treated samples. Further analysis revealed that the toxicity effect of 3-HP was due to the cation and anion components of this acid and some effects-specific to 3-HP. Genes related to the oxidative stress, DNA protection, and repair were upregulated in treated cells due to the lowered cytoplasmic pH caused by accumulated cations. 3-HP-treated E. coli used the arginine acid tolerance mechanism to increase the cytoplasmic pH. Additionally, the anion effects were manifested as imbalance in the osmotic pressure. Analysis of top ten highly upregulated genes suggests the formation of 3-hydroxypropionaldehyde under 3-HP stress. The transcriptomic analysis shed light on the global genetic reprogramming due to 3-HP stress and suggests strategies for increasing the tolerance of E. coli toward 3-HP.