G-ABCD is a biosimilar product of amphotericin B colloidal dispersion (ABCD). This study was designed to systematically examine the plasma pharmacokinetics (PK) and tissue distribution of G-ABCD in rats, using amphotericin B deoxycholate (DAmB) as a positive control. Male Sprague-Dawley rats received single dose or 14 doses of G-ABCD (1.0, 2.0 or 5.0 mg/kg) or the conventional micellar formulation DAmB) (1.0 mg/kg) via intravenous injection. Plasma and tissue samples were obtained for analysis of amphotericin B concentration by liquid chromatography-tandem mass spectrometry. After a single-dose administration of 1 mg/kg, G-ABCD resulted in a significantly lower plasma peak drug concentration (Cmax) (1536 vs. 5256 ng/mL) and area under the curve from time 0 to ∞ (AUC0-∞) (3972 vs. 7006 h ng/mL) of non-complexed amphotericin B than DAmB. G-ABCD was associated with quicker distribution but slower elimination of amphotericin B than DAmB. Amphotericin B concentration reached a steady state after seven doses of G-ABCD. After multiple doses of 1 mg/kg, G-ABCD showed a lower peak level and longer half-life of amphotericin B in plasma than DAmB. G-ABCD treatment in rats was associated with relatively higher distribution to liver and spleen, but reduced amphotericin B delivery to kidneys, the major target organ of toxicity. These results suggest that G-ABCD provides a flatter but more lasting plasma level of amphotericin B and lower kidney burden in rats than DAmB.