Hypothesis Central Gαi2-subunit proteins mediate the centrally evoked sympathoinhibitory, natriuretic and diuretic responses to acute i.v. isovolumetric sodium loading in conscious rats. Methods An isovolumetric sodium load (1M NaCl) was administered over 2h by i.v. infusion (20 μl/min) in conscious naïve or chronic bilateral renal denervated (RDNX) Sprague-Dawley rats pre-treated intracerebroventricularly (i.c.v.; 24-h) with a scrambled (SCR) or Gαi2 oligodeoxynucleotide (ODN; 25 μg/each; N=6/group). MAP, HR and urine output were recorded for 5-hrs (1hr control, 2h NaCl load, 2 h recovery). Plasma norepinephrine (NE) content and plasma renin activity (PRA) were measured during control, sodium load and recovery periods. Results In response to an i.v. isovolumetric 1M sodium load, prior down-regulation of brain Gαi2-proteins attenuated the natriuresis (peak ΔUNaV [μeq/μl]; SCR 25±3 vs. Gαi2 ODN 12±2, P<0.05) and diuresis (peak ΔUV [μl/min]; SCR 53±5 vs. Gαi2 ODN 24±3, P<0.05), and abolished the global sympathoinhibitory response (peak Δplasma NE [% control]; SCR −78±5 vs. Gαi2 ODN −3±2, P<0.05) without altering the suppression of PRA activity (peak ΔPRA activity [Ang I generation ng/ml/h]; SCR −2.6±0.3 vs. Gαi2 ODN −2.4±0.2). In contrast, in chronic RDNX rats, Gαi2 ODN pre-treatment failed to alter the natriuretic (peak ΔUNaV [μeq/μl]; SCR 20±2 vs. Gαi2 ODN 21±2) and diuretic responses (peak ΔUV [μl/min]; SCR 45±4 vs. Gαi2 ODN 44±3) and partially restored the global sympathoinhibitory response to the sodium load (peak Δplasma NE [% control]; SCR −74±6vs. Gαi2 ODN −44±4, P<0.05). Conclusion We have discovered a novel brain Gαi2-protein mediated sympathoinhibitory renal nerve dependent (renin-angiotensin system independent) pathway that has a critical role in the endogenous central neural mechanisms activated to maintain fluid and electrolyte homeostasis. AHA 2250585, NIH P20RR018766.