The nucleus accumbens (NAc) is a critical brain reward region that mediates the rewarding effects of drugs of abuse, including those of morphine and other opiates. Drugs of abuse induce widespread alterations in gene transcription and dendritic spine morphology in medium spiny neurons (MSNs) of the NAc that ultimately influence NAc excitability and hence reward‐related behavioral responses. Growing evidence indicates that within the NAc small GTPases are common intracellular targets of drugs of abuse where these molecules regulate drug‐mediated transcriptional and spine morphogenic effects. The RhoA small GTPase is among the most well‐characterized members of the Ras superfamily of small GTPases, and recent work highlights an important role for hippocampal RhoA in morphine‐facilitated reward behavior. Despite this, it remains unclear how RhoA pathway signaling in the NAc is affected by withdrawal from morphine. To investigate this question, using subcellular fractionation and subsequent protein profiling we examined the expression of key components of the RhoA pathway in NAc nuclear, cytoplasmic, and synaptosomal compartments during multiple withdrawal periods from repeated morphine administration. Furthermore, using in vivo viral‐mediated gene transfer, we determined the consequences of revealed RhoA pathway alterations on NAc MSN dendritic spine morphology. Our findings reveal an important role for RhoA signaling cascades in mediating the effects of long‐term morphine withdrawal on NAc MSN dendritic spine elimination. Open science badges This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. The expression profile of the RhoA network was examined in nuclear, cytosolic, and synaptosomal fractions from nucleus accumbens tissue following multiple withdrawal periods from repeated morphine administration. We found that short‐term morphine withdrawal increases the expression of a key component of the RhoA network in nuclear fractions, and this increase is directed toward specific actin cytoskeleton states. Long‐term morphine withdrawal, on the other hand, increases expression of a component of the RhoA network in synaptosomal fractions. These findings have implications for understanding the mechanisms that contribute to drug‐seeking behavior during opiate withdrawal. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/