In Klebsiella pneumoniae, 3-hydroxypropaldehyde is converted to 3-hydroxypropionic acid (3-HP) by aldehyde dehydrogenase (ALDH) with NAD+ as a cofactor. Although ALDH overexpression stimulates the formation of 3-HP, it ceases to accumulate when NAD+ is exhausted. Here we show that NAD+ regeneration, together with ALDH overexpression, facilitates 3-HP production and benefits cell growth. Three distinct NAD+-regenerating enzymes: NADH oxidase and NADH dehydrogenase from K. pneumoniae, and glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae, were individually expressed in K. pneumoniae. In vitro assay showed their higher activities than that of the control, indicating their capacities to regenerate NAD+. When they were respectively co-expressed with ALD4, an ALDH from S. cerevisiae, the activities of ALD4 were significantly elevated compared with that expressing ALD4 alone, suggesting that the regenerated NAD+ enhanced the activity of ALD4. More interestingly, the growth rates of all NAD+-regenerating strains were prolonged in comparison with the control, indicating that NAD+ regeneration stimulated cell proliferation. This study not only reveals the reliance of ALD4 activity on NAD+ availability but also provides a method for regulating the dha regulon.