To characterize the pathway of ceftriaxone penetration into intervertebral discs, rat-tail discs were collected and immersed in a ceftriaxone solution for an in vitro study. For this in vivo experiment, plasma and tail discs were collected 30 min after ceftriaxone administration (463.2 mg/kg, equal to 500 mg/kg disodium salt, iv). The ceftriaxone levels in the plasma and discs were determined with HPLC by a reversed-phase C18 column and a mobile phase of methanol and a 10 mM K2HPO4 solution (pH 6.7) (21:79, v/v). The results indicate that ceftriaxone was undetectable by the in vitro model. The concentrations of ceftriaxone in plasma and in intervertebral discs were 147.4 ± 21.2 and 4.0 ± 1.7 mg/mL (as disodium salt), respectively. Our results suggest that a sufficient blood supply is required for the passive diffusion of ceftriaxone into intervertebral discs. The concentration ratio of ceftriaxone in intervertebral disc-to-plasma was about 2.7% at 30 min after ceftriaxone disodium administration (500 mg/kg, iv).