An aberrant pro-inflammatory microglia response has been associated with most neurodegenerative disorders. Identifying microglia druggable checkpoints to restore their physiological functions is an emerging challenge. Recent data have shown that microglia produce de novo neurosteroids, endogenous molecules exerting potent anti-inflammatory activity. Here, the role of neurosteroidogenesis in the modulation of microgliosis was explored in human microglia cells. In particular, CYP11A1 inhibition or TSPO pharmacological stimulation, crucial proteins involved in the rate limiting step of the neurosteroidogenic cascade, were employed. CYP11A1 inhibition led microglia to acquire a dysfunctional and hyperreactive phenotype, while selective TSPO ligands promoted the establishment of an anti-inflammatory one. Analysis of specific neurosteroid levels (neurosteroidome) identified allopregnanolone/pregnanolone as crucial metabolites allowing controlled activation of microglia. Importantly, the neurosteroid shift towards a greater androgenic/estrogenic profile supported the transition from pro-inflammatory to neuroprotective microglia, suggesting the therapeutic potential of de novo microglial neurosteroidogenesis stimulation for neuroinflammatory-related disorders.