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Inhibition of the glucagon receptor (GCGR) has been identified as a potential therapeutic approach for the treatment of type 2 diabetes. However, a small molecule drug candidate antagonizing GCGR (BAY16) failed during preclinical drug development, in part due to drug induced hepatotoxicity in animals. Since there is evidence to suggest that endogenous GCGR signaling might be important for hepatocyte survival, we hypothesized that on-target effects, i.e., modulation of GCGR activity by BAY16, may contribute to BAY16 hepatotoxicity and associated gene expression changes in rats. To understand the role of GCGR inhibition in BAY16 toxicity, we analyzed cell viability and gene expression profiles in non-silenced and GCGR-targeting siRNA transfected primary rat hepatocytes with and without exposure to BAY16 to discriminate between on- and off-target effects of BAY16. siRNA-mediated silencing of the GCGR did not affect cell viability in primary rat hepatocytes, indicating that cytotoxicity of BAY16 occurs independent of its pharmacological effects. In support of this, gene expression analysis of GCGR silenced hepatocytes revealed no transcriptional alterations relevant to toxicity. In contrast, BAY16 caused a concentration-dependent decrease in cell viability, along with changes in the expression of genes associated with altered xenobiotic metabolism, oxidative stress, increased fatty acid synthesis, and alterations in cholesterol and bile acid metabolic processes. Based on gene expression data, it appears that hepatocytes inhibit cholesterol synthesis and increase detoxifying and eliminating processes in order to protect themselves from accumulation of bile acids, cholesterol or drug intermediates. Importantly, comparison of transcriptional changes in the absence and presence of GCGR revealed that the same pathways were affected in both silenced and non-silenced hepatocytes, indicating that BAY16 toxicity occurs independent of the GCGR receptor.