Objective Glycerol-based biosynthesis of 3-hydroxypropionic acid (3-HP) inKlebsiella pneumoniaeinvolves two reactions: glycerol conversion to 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase, and 3-HPA conversion to 3-HP by aldehyde dehydrogenase (ALDH). The ALDH catalysis consumes a lot of cofactor nicotinamide adenine dinucleotide (NAD(+)), which constrains 3-HP production. Results Here we report that intensifying niacin-based biosynthesis of NAD(+)can substantially enhance 3-HP production. We constructedtacpromoter-driven NAD(+)synthesis pathway inK. pneumoniae. The strain only overexpressing nicotinate phosphoribosyltransferase (PncB) showed 14.24% increase in the production of NAD(+)relative to the stain harboring an empty vector. When PncB was coexpressed with PuuC (one of native ALDHs), the recombinant strain exhibited increased ALDH activity but slightly reduced 3-HP production due to plasmid burden. When 30 mg niacin l(-1)(a substrate for biosynthesis of NAD(+)) was added into shake flask, the strain produced 0.55 g 3-HP l(-1), which was 2.75 times that of the control. In a 5-L bioreactor, replenishment of niacin led to 36.43% increase of 3-HP production. Conclusions These results indicated that intensifying niacin-based biosynthesis of NAD(+)boosts 3-HP production.