Application of omics-based technologies is a widely used approach in research aiming to improve testing strategies for human health risk assessment. In most of these studies, however, temporal variations in gene expression caused by the circadian clock are a commonly neglected pitfall. In the present study, we investigated the impact of the circadian clock on the response of the hepatic transcriptome after exposure of mice to the chemotherapeutic agent cyclophosphamide (CP). Analysis of the data without considering clock progression revealed common responses in terms of regulated pathways between light and dark phase exposure, including DNA damage, oxidative stress, and a general immune response. The overall response, however, was stronger in mice exposed during the day. Use of time-matched controls, thereby eliminating non-CP-responsive circadian clock-controlled genes, showed that this difference in response was actually even more pronounced: CP-related responses were only identified in mice exposed during the day. Only minor differences were found in acute toxicity pathways, namely lymphocyte counts and kidney weights, indicating that gene expression is subject to time of day effects. This study is the first to highlight the impact of the circadian clock on the identification of toxic responses by omics approaches.