Homeostasis of inorganic phosphate in the human body is maintained by regulated absorption, metabolism, and excretion. Sodium-dependent phosphate transporters (NaPi) mediate the transport of inorganic phosphate (P(i)) in cells in response to dietary phosphate consumption, hormones, and growth factors. NaPi2b is a member of the sodium-dependent phosphate transporter family, with a distinct pattern of expression and regulation. Signaling pathways activated by mitogens, glucocorticoids, and metabolic factors have been implicated in regulating P(i) transport via NaPi2b. Inactivation of NaPi2b function by mutations has been linked to human pathologies, such as pulmonary alveolar microlithiasis. In this study, we describe the generation and characterization of monoclonal antibodies against human NaPi2b. The monoclonal antibodies were shown to recognize specifically transiently overexpressed and endogenous NaPi2b in commonly used immunoassays, including Western blotting, immunoprecipitation, and immunohistochemistry. These properties make them particularly valuable reagents for elucidating NaPi2b function in health and disease.