Nitric oxide (NO) antagonizes hepatic stellate cell (HSC) contraction. However, in cirrhosis, activated HSC demonstrate impaired responses to NO. The defects that cause impaired responses to NO in activated HSC as well as the broader mechanisms by which NO exerts its effects in activated HSC remain incompletely understood. In HSC isolated from normal rats, the NO donor DEA NONOate (DEAN), significantly increased cGMP production, and reduced serum-induced contractile responses by 25%. The soluble guanylate cyclase (sGC) inhibitor, ODQ, abolished 50% of DEAN mediated effects while the soluble cGMP analogue 8-Br-cGMP, provided 50% of the observed response of DEAN, suggesting both cGMP-protein kinase G (PKG)-dependent and independent mechanisms of NO-mediated antagonism of contraction in normal HSC. However, NO donors did not increase cGMP production from in vivo activated HSC isolated from bile duct ligated (BDL) rats suggesting a defect in the cGMP-dependent effector pathway. These changes were also associated with alterations in intracellular Ca²⁺ accumulation in HSC from BDL rats. More detailed molecular studies were pursued in the LX-2 cell line, which also demonstrated a lack of cGMP generation in response to NO donors and also showed a lack of effect of ODQ and 8-Br-cGMP in modulation of the NO response. However, the cGMP independent effects observed in response to exogenous NO was maintained in LX-2 and was associated with increased NO induced S-nitrosylation of cellular proteins; an effect that was reiterated in primary hepatic stellate cells. Additionally, adenoviral-based overexpression of PKG significantly attenuated the contraction of LX-2 by 25% in response to 8-Br-cGMP. In summary, these studies demonstrate that NO antagonism of HSC contractility occurs through cGMP-dependent and independent pathways. The activation process in HSC is associated with maintenance of cGMP-independent actions of NO, but defects in cGMP-PKG-dependent NO signaling, the latter of which can be improved by PKG gene delivery in LX-2 cells. Activation of targets downstream from NO-cGMP in activated HSC may represent a novel therapeutic target for portal hypertension.