Germline mutations in PTEN account for ~10% of cases of autism spectrum disorder (ASD) with coincident macrocephaly. To explore the importance of nuclear PTEN in the development of ASD and macrocephaly, we previously generated a mouse model with predominantly cytoplasmic localization of Pten (Pten).Cytoplasmic predominant Pten localization results in a phenotype of extreme macrocephaly and autistic-like traits. Transcriptomic analysis of the Pten cortex found upregulated gene pathways related to myeloid cell activation, myeloid cell migration, and phagocytosis. These transcriptomic findings were used to direct in vitro assays on Pten wild-type and Pten microglia. We found increased Iba1 and C1q expression with enhanced phagocytic capacity in Pten microglia, indicating microglial activation. Moreover, through a series of neuron-microglia co-culture experiments, we found Pten microglia are more efficient at synaptic pruning compared with wild-type controls. In addition, we found evidence for neuron-microglia cross-talk, where Pten neurons elicit enhanced pruning from innately activated microglia. Subsequent in vivo studies validated our in vitro findings. We observed a concurrent decline in the expression of Pten and synaptic markers in the Pten cortex. At ~3 weeks of age, with a 50% drop in Pten expression compared with wild-type levels, we observed enhanced activation of microglia in the Pten brain. Collectively, our data provide evidence that dysregulated Pten in microglia has an etiological role in microglial activation, phagocytosis, and synaptic pruning, creating avenues for future studies on the importance of PTEN in maintaining microglia homeostasis.