The aim of this study was to analyze the photostability and phototoxicity mechanism of anthracene (ANT) in a human skin epidermal cell line (HaCaT) at ambient environmental intensities of sunlight/UV-R (UV-A and UV-B). Photomodification of ANT under sunlight/UV-R exposure produced two photoproducts, anthrone and 9,10 anthracenedione. Generation of 1O2, O2•− and •OH was measured under UV-R/sunlight exposure. Involvement of reactive oxygen species (ROS) was further substantiated by their quenching with free radical quenchers. Photodegradation of 2-deoxyguanosine and linoleic acid peroxidation showed that ROS were mainly responsible for ANT phototoxicity. ANT generates significant amount of intracellular ROS in cell line. Maximum cell viability (85%) was reduced under sunlight exposure (30 min). Results of MTT assay accord NRU assay. ANT (0.01 μg mL−1) induced cell-cycle arrest at G1 phase. RT-PCR demonstrated constitutive inducible mRNA expression of CYP 1A1 and 1B1 genes. Photosensitive ANT upregulates CYP 1A1 (2.2-folds) and 1B1 (4.1-folds) genes. Thus, the study suggests that ROS and DNA damage were mainly responsible for ANT phototoxicity. ANT exposure may be deleterious to human health at ambient environmental intensities reaching the earth’s surface through sunlight.