We examined whether pretreatment of mouse brain blood vessel endothelial cell clone 4 (MBEC4) cells with sodium nitroprusside (SNP), a $NO_x$ donor, as an in vitro model of the blood-brain barrier could affect P-glycoprotein (P-gp) functional activity. Uptake into the cells and MBEC4 plasma membrane vesicles (MPMVs) in the presence or absence of SNP pretreatment was used to investigate functional changes. Increased accumulation of $[^3H]$vincristine, a widely used substrate for P-gp, into MBEC4 was observed upon SNP pretreatment, likely due to impaired P-gp function. To better understand the mechanism of the impairment, MPMVs were prepared and characterized in terms of purity and $Na^+$-dependent glucose uptake. $[^3H]$daunomycin uptake into MPMVs was diminished after SNP pretreatment in the presence of an ATP-regenerating system, indicating that the functional activity of P-gp was impaired after exposure to SNP. Under conditions of excess ATP, daunomycin uptake into the vesicles was still decreased after SNP pretreatment, indicating that SNP interacted directly with the transport system, but not with the ATP-regenerating system. Together, these results suggest that NO or $NO_x$ functionally impairs P-gp in the in vitro blood-brain barrier model with SNP pretreatment.