Opiates addiction is a major public health problem worldwide. This condition has been conventionally considered to be a consequence of the neuronal activation of classical opioid receptors. However, nowadays numerous studies point out to the TLR4 signaling pathway as a target for opiates and its participation, through the modulation of neuroinflammatory mechanisms, in many of these drugs’ effects has been postulated. Thus, glial cells are currently the focus of a number of studies in order to understand their contribution to the addictive properties of opiates, as they have the ability to modulate synaptic plasticity. In our study, proteins levels of TLR4, JNK, P38, and ERK, both in their phosphorylated and total forms, were quantified via western blot after acute and chronic morphine treatment as well as after morphine withdrawal, in the prefrontal cortex (PFC), that mediates in executive function and emotional control, of rats. Additionally, qPCR was used to study the expression of glial activity markers. Our results show that acute morphine administration decreases the expression of TLR4, consequently reducing the activity of some of the TLR4-regulated kinases, and the mRNA expression levels of Iba1, among other neuroinflammatory mediators, in the PFC. Likewise, chronic morphine treatment was observed to diminish TLR4 and some glial markers expression in this brain area. Our findings provide evidence of differential expression and activity of synaptic molecules after opioid treatment in the PFC that will likely contribute to the development of new therapeutic strategies aimed at glial cells for the treatment of opiates use disorders.