ABSTRACT Previously, we characterized a pathway necessary for the processing of NAD ⁺ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae . Here we report on the role of NadR, which is essential for NAD ⁺ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV ⁺ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae . Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD ⁺ under in vitro conditions and by NAD ⁺ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD ⁺ synthesis and is also involved in controlling the NAD ⁺ biosynthesis rate.