Binding of angiotensin II (Ang II) to the AT1 receptor (AT1R) in the proximal tubule stimulates NHE3 activity through multiple signaling pathways. However, the effects of Ang II/AT1R-induced Gi activation and subsequent decrease in cAMP accumulation on NHE3 regulation are not well established. We therefore tested the hypothesis that Ang II reduces cAMP/PKA-mediated phosphorylation of NHE3 on serine 552 and, in doing so, stimulates NHE3 activity. Under basal conditions, Ang II stimulated NHE3 activity but did not affect PKA-mediated NHE3 phosphorylation at serine 552 in opossum kidney (OKP) cells. However, in the presence of cAMP-elevating agent forskolin (FSK), Ang II blocked FSK-induced NHE3 inhibition, reduced intracellular cAMP concentrations, lowered PKA activity and prevented the FSK-mediated increase in NHE3 serine 552 phosphorylation. All effects of Ang II were blocked by pretreating OKP cells with the AT1R antagonist losartan, highlighting the contribution of the AT1R/Gi pathway in Ang II-mediated NHE3 upregulation under cAMP-elevating conditions. Accordingly, Gi inhibition by pertussis toxin treatment decreased NHE3 activity both in vitro and in vivo and, more importantly, prevented the stimulatory effect of Ang II on NHE3 activity in rat proximal tubules. Collectively, our results suggest that Ang II counteracts the effects of cAMP/PKA on NHE3 phosphorylation and inhibition by activating the AT1R/Gi pathway. Moreover, these findings support the notion that NHE3 dephosphorylation at serine 552 may represent a key event in the regulation of renal proximal tubule sodium handling by Ang II in the presence of natriuretic hormones that promote cAMP accumulation and transporter phosphorylation.