Multiple isoforms of inhibitory Gα-subunits (Gα_i1,2,3, as well as Gα_o) are present within the heart, and their role in modulating pacemaker function remains unresolved. Do inhibitory Gα-subunits selectively modulate parasympathetic heart rate responses? Published findings using a variety of experimental approaches have implicated roles for Gα_i2, Gα_i3, and Gα_o in parasympathetic signal transduction. We have compared in vivo different groups of mice with global genetic deletion of Gi_α1/Gα_i3, Gα_i2, and Gα_o against littermate controls using implanted ECG telemetry. Significant resting tachycardia was observed in Gα_i2^−/− and Gα_o^−/− mice compared with control and Gα_i1^−/−/Gα_i3^−/− mice (P < 0.05). Loss of diurnal heart rate variation was seen exclusively in Gα_o^−/− mice. Using heart rate variability (HRV) analysis, compared with littermate controls (4.02 ms² ± 1.17; n = 6, Gα_i2^−/−) mice have a selective attenuation of high-frequency (HF) power (0.73 ms² ± 0.31; n = 5, P < 0.05). Gα_i1^−/−/Gα_i3^−/− and Gα_o^−/− cohorts have nonsignificant changes in HF power. Gα_o^−/− mice have a different basal HRV signature. The observed HRV phenotype in Gα_i2^−/− mice was qualitatively similar to atropine (1 mg/kg)-treated controls [and mice treated with the GIRK channel blocker tertiapinQ (0.05 mg/kg)]. Maximal cardioinhibitory response to the M₂-receptor agonist carbachol (0.5 mg/kg) compared with basal heart rate was attenuated in Gα_i2^−/− mice (0.08 ± 0.04; n = 6) compared to control (0.27 ± 0.04; n = 7 P < 0.05). Our data suggest a selective defect of parasympathetic heart rate modulation in mice with Gα_i2 deletion. Mice with Gα_o deletion also have a defect in short-term heart rate dynamics, but this is qualitatively different to the effects of atropine, tertiapinQ, and Gα_i2 deletion. In contrast, Gα_i1 and Gα_i3 do not appear to be essential for parasympathetic responses in vivo.