This study examined the impact of using cultural materials on listening comprehension among male and female Iranian EFL learners. To this end, 96 upper-intermediate male (n = 48) and female (n = 48) EFL learners out of 130, were chosen through administering an Oxford Quick Placement Test (OQPT). The participants of each gender were randomly assigned into four equal groups; group A (Target Culture = TC), group B (International Target Culture = ITC), group C (Source Culture = SC) and group D (Culture-Free = CF). Then, a listening comprehension pre-test was administered to assess the participants' listening comprehension at the beginning of the course. After the pre-test, the researcher practiced the treatment on the experimental groups. Each group received listening comprehension materials that reflected a particular culture. During the treatment, some audio files related to American and English culture (for group A), culture of different foreign countries such as Japan, Australia and France (for group B), Persian culture (group C), and culture free materials (for group D), were chosen. Finally, a post-test of listening comprehension was employed after the treatment to gather sufficient data. Two-way ANOVA was run to analyze the data among all groups' pre and post-tests regarding the male and female participants. The findings proposed that the participants performed differently on the post-test showing that culturally oriented language materials enhance the Iranian EFL learners' listening comprehension. Furthermore, the results indicated that the male learners did better than female ones on the post-test.