The article presents the results of a cross-cultural affective images perception study by Americans and Russians and reveals the degree of cultural factor influence on the stimuli assessment by American and Russian men and women. The hypothesis is that assessments of affective images by American and Russian respondents will have statistical differences due to the linguistic and cultural specificity of the ethnic groups; it is also assumed there are cross-cultural gender differences in the assessment. The study used the method of psycholinguistic questioning with seven-point scaling. 84 images from the open American database of affective images (“Open Affective Standardized Image Set”) were used as research material. The respondents were 34 men and 58 women. The results of the analysis did not show significant cross-cultural differences in ratings of affective images with reference to valence type or emotional evaluation/response. In general, Americans and Russians had a similar distribution of image ratings. However, a statistically significant difference has been found in the ratings of images with different valence types (P < 0.001). Negative and positive images were rated higher by Russians in terms of emotional evaluation, in contrast to Americans, most of whose emotional responses had neutral ratings. There was also a statistically significant difference in the ratings of different thematic images (P < 0.05). Nature images were rated by Russians as causing a feeling of comfort, while Americans noted their neutral impact on them. Images of objects, on the contrary, received the opposite ratings from the respondents. Moreover, cross-cultural gender differences have been revealed between Russian and American women in image ratings based on emotional evaluation and valence parameters (P < 0.05). Russian women rated most of the images as having a positive or negative impact, while the majority of American women’s ratings tended to be neutral. This confirms the influence of the emotional stimulus, valence type, image theme, as well as gender factor on the processing of emotionally coloured units by representatives of different cultures.