Recent studies suggest that botulinum toxin type A (BT-A) might inhibit the release of pain neurotransmitters from central primary afferent terminals, including glutamate. Previously we have demonstrated that peripheral BT-A and intrathecal NMDA antagonist (AP5) have an additive antinociceptive effect in combination. The aim of the present study was to investigate potential changes in the expression of spinal glutamate receptors and a membrane associated scaffolding protein in neural postsynaptic densities PSD-95, as markers of central sensitization involved in generation of pathological pain. Ipsilateral dorsal horns from naïve and 5% formalin (20 μL s.c. into the hind paw) treated male Wistar rats were collected and homogenized to analyze the expression of: a) NR1 and b) NR1 phospho-Ser897 subunits of NMDA receptor; c) GluR1 and d) GluR1 phospho-Ser845 subunits of AMPA receptor; and e) PSD-95 by western blot. Formalin-treated groups received saline or BT-A (5 U/kg, s.c. into the hind paw) 5 days, and/or AP-5 (10 μg/10 μL, intrathecal) 10 min before the test. Experiments were approved by Croatian Ministry of Agriculture Veterinary and Food Safety Directorate (permit no. EP 03-2/2015). Compared to naïve animals, the expression of glutamate receptors and their phosphorylated forms was increased in formalin-treated rats, which is in line with the induction of inflammatory pain. However, no changes were observed following the application of BT-A, AP5 or their combination. PSD-95 expression was unchanged across all groups. Therefore, the antinociceptive effect of BT-A and/or AP-5 was not accompanied by the expected reduction in selected central sensitization markers’ expression. Considering that BT-A’s mechanism of action includes the inhibition of neurotransmitters’ release, the consequent reduction of glutamate receptors may not be visible in acute pain model and should be investigated further in chronic pain model, together with the more probable effect on spinal glutamate level.