The incidences of skin cancers resulting from chronic ultraviolet radiation (UVR) exposure are on the incline in both Australia and globally. Hence, the cellular and molecular pathways that are associated with UVR-induced photocarcinogenesis need to be urgently elucidated, in order to develop more robust preventative and treatment strategies against skin cancers. In vitro investigations into the effects of UVR (in particular, the highly mutagenic UVB wavelength) have, to date, mainly involved the use of cell culture and animal models. However, these models possess biological disparities to native skin, which, to some extent, have limited their relevance to the in vivo situation. To address this, we characterized a three-dimensional, tissue-engineered human skin equivalent (HSE) model (consisting of primary human keratinocytes cultured on a dermal-derived scaffold) as a representation of a more physiologically relevant platform to study keratinocyte responses to UVB. Significantly, we demonstrate that this model retains several important epidermal properties of native skin. Moreover, UVB irradiation of the HSE constructs was shown to induce key markers of photodamage in the HSE keratinocytes, including the formation of cyclobutane pyrimidine dimers, the activation of apoptotic pathways, the accumulation of p53, and the secretion of inflammatory cytokines. Importantly, we also demonstrate that the UVB-exposed HSE constructs retain the capacity for epidermal repair and regeneration after photodamage. Together, our results demonstrate the potential of this skin equivalent model as a tool to study various aspects of the acute responses of human keratinocytes to UVB radiation damage.