Bitewing radiography is an important modality useful for the evaluation of teeth in patients of various ages and in different stages of tooth eruption. Clinical examination of proximal surfaces for caries may result in false negative results, especially in tight contact areas. Thus, radiography, as an adjunct to clinical examination, is used as a routine diagnostic modality for caries detection. The objective of this study was to assess the in vitro effect of changing the horizontal angulation of X-ray beam on the detection of proximal enamel caries in bitewing radiographs. This in vitro study was conducted on 150 caries-free human premolars (code: p/16/35/9/210). The teeth were randomly divided into 3 groups (n = 50). Group 1 served as the control group and no carious lesions were induced in this group. Teeth in groups 2 and 3 were immersed in demineralizing solution for 2 and 4 months, respectively. After induction of caries and its radiographic confirmation, the teeth were mounted in wax in groups of 3 and bitewing radiographs were obtained at 0°, 5°, 10° and 15° horizontal angles. The sensitivity values of bitewing radiographs at 0°, 5°, 10° and 15° horizontal angles were 88%, 90%, 88%, 92% in group 2 and 88%, 94%, 94% and 94% in group 3, respectively. The specificity values of bitewing radiographs at 0°, 5°, 10° and 15° horizontal angles were 92%, 86%, 84% and 76%. The accuracy of bitewing radiographs at 0°, 5°, 10° and 15° horizontal angles was 89.3%, 90%, 88.7% and 87.3%, respectively. The highest diagnostic accuracy was obtained at 5° horizontal angle for caries detection; however, the difference in this regard among the tested horizontal angles was not statically significant (p = 0.846). Based on the results of this study, changing the horizontal angulation has no significant effect on the detection of proximal enamel caries in bitewing radiographs.