Glutamate transporters (GluTs) are important for maintaining optimal glutamate concentrations at the synapse. This allows proper synaptic response, plasticity and prevents neurotoxicity. It has been shown that the β-lactam antibiotic ceftriaxone (Rocephin) induces an up-regulation of the glutamate transporter GLT-1. This GLT-1 up-regulation blocks the metabotropic glutamate receptor (mGluR) dependent long-term depression (LTD) at the mossy fiber (MF)-CA3 hippocampal synapse. It also has negative effects on long-term potentiation (LTP). However, the effects of GLT-1 up-regulation on hippocampal learning in rats are not known. In this study, we examine the role of chronic administration of ceftriaxone on novel object recognition, which is a hippocampal-dependent spatial learning task. Male Sprague Dawley rats (2–3 months old) were administered ceftriaxone (via i.p. injections, 200 mg/kg) for 8 consecutive days prior to training and testing on a standard novel object recognition task. We found that rats administered with ceftriaxone display memory impairments in novel object recognition, when compared to control rats (p < 0.05). Our findings show that a potential up-regulation of GLT-1 via ceftriaxone administration has detrimental effects on spatial learning and memory in rats. Our results further support the notion that glutamate transporters provide an essential regulatory role in hippocampal learning and memory.