The use of acetate as carbon feedstock can enhance sustainability and economics of the current bio-productions. This study explored the potential of acetate for the production of 3-hydroxypropionic acid by engineered Pseudomonas denitrificans. Heterologous mcr (encoding malonyl-CoA reductase) from Chloroflexus aurantiacus and endogenous accABCD (encoding acetyl-CoA carboxylase) were overexpressed in P. denitrificans. Carbon flux to 3-HP synthesis at the malonyl-CoA node was promoted by suppressing fatty acid synthesis through addition of cerulenin or deletion of fabF gene. In addition, stimulation of glyoxylate shunt and/or TCA cycle were attempted. Recombinant P. denitrificans overexpressing mcr and accABCD produced 19.3 mM 3-HP with cerulenin addition, and 14.2 mM with fabF deletion, respectively. Furthermore, the non-growing cells devoid of fabF could continuously produce 3-HP up to 40.4 mM without losing its production activity for 22 h. This study demonstrates that acetate is a good substrate for 3-HP production by recombinant P. denitrificans.