3-Hydroxypropionic acid (3-HP) is an important platform chemical that can be used to synthesize a range of chemical compounds. A previous study demonstrated that recombinant Escherichia coli stains can produce 3-HP from glycerol in the presence of vitamin B₁₂ (coenzyme B₁₂), when overexpressed with a coenzyme B₁₂-dependent glycerol dehydratase (DhaB) and an aldehyde dehydrogenase. The present study examined the production of 3-HP in recombinant Klebsiella pneumoniae strains, which naturally synthesizes vitamin B₁₂ and does not require supplementation of the expensive vitamin. The NAD⁺-dependent gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae alone or with its DhaB was overexpressed homologously, and two major oxidoreductases, DhaT and YqhD, were disrupted. Without vitamin B₁₂ addition, the recombinant K. pneumoniae ΔdhaTΔyqhD overexpressing PuuC could produce ∼3.8 g/L 3-HP in 12 h of flask culture. However, this was possible only under the appropriate aeration conditions; 1,3-propanediol (1,3-PDO) (instead of 3-HP) was mainly produced when aeration was insufficient, whereas a very small amount of both 3-HP and 1,3-PDO were produced when aeration was too high. The production of a small amount of 3-HP under improper aeration conditions was attributed to either slow NAD⁺ regeneration (under low aeration) or reduced vitamin B₁₂ synthesis (under high aeration). In a glycerol fed-batch bioreactor experiment under a constant DO of 5%, the strain, K. pneumoniae ΔdhaTΔyqhD, overexpressing both PuuC and DhaB could produce >28 g/L 3-HP in 48 h with a yield of >40% on glycerol. Only small amount of 3-HP was produced when cultivation was carried out at a constant aeration of 1 vvm or constant 10% DO. These results show that K. pneumoniae is potentially useful for the production of 3-HP in an economical culture medium that does not require vitamin B₁₂. The results also suggest that the aeration conditions should be optimized carefully for the efficient production of 3-HP while using this strain.