Nitric oxide (NO) can directly modulate cardiac contractility by accelerating relaxation and reducing diastolic tone. The intracellular mechanisms underlying these contractile effects are poorly understood. Here we investigate the role of cyclic GMP‐dependent protein kinase (PKG) in the contractile response to exogenous NO in rat ventricular myocytes. Isolated ventricular myocytes were stimulated electrically and contractility was assessed by measuring cell shortening. Some cells were loaded with the fluorescent Ca²⁺ probe indo‐1 AM for simultaneous assessment of the intracellular Ca²⁺ transient. The NO donor diethylamine NONOate (DEA/NO, 10 μm) significantly increased resting cell length, reduced twitch amplitude and accelerated time to 50 % relaxation (to 100.8 ± 0.2, 83.7 ± 3.0 and 88.9 ± 3.7 % of control values, respectively). The contractile effects of DEA/NO occurred without significant changes in the amplitude or kinetics of the intracellular Ca²⁺ transient, suggesting that the myofilament response to Ca²⁺ was reduced. These effects were abolished by inhibition of either guanylyl cyclase (with 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one; ODQ, 10 μm) or PKG (with Rp‐8‐Br‐cGMPs, 10 μm) suggesting that, at the concentration investigated, the effects of DEA/NO were mediated exclusively by PKG, following activation of guanylyl cyclase and elevation of cGMP. Direct activation of PKG with 8‐pCPT‐cGMP (10 μm) mimicked the effects of DEA/NO (resting cell length and time to 50 % relaxation were 100.6 ± 0.1 and 90.5 ± 1.5 % of control values, respectively).The reduced myofilament Ca²⁺ responsiveness was not attributable to an intracellular acidosis since the small reduction in pH_i induced by DEA/NO was found to be uncoupled from its contractile effects. However, hearts treated with DEA/NO (10 μm) showed a significant increase (1.4‐fold; P < 0.01) in troponin I phosphorylation compared to control, untreated hearts. These results suggest that the reduction in myofilament Ca²⁺ responsiveness produced by DEA/NO results from phosphorylation of troponin I by PKG.