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<i>Culex quinquefasciatus</i>, one of the most significant mosquito vectors in the world, is widespread in most parts of southern China. A variety of diseases including Bancroft’s filariasis, West Nile disease, and St. Louis encephalitis could be transmitted by the vector. Mosquitoes have been shown to host diverse bacterial communities that vary depending on environmental factors such as temperature and rainfall. In this work, 16S rDNA sequencing was used to analyze the seasonal variation of midgut bacterial diversity of <i>Cx. Quinquefasciatus</i> in Haikou City, Hainan Province, China. Proteobacteria was the dominant phylum, accounting for 79.7% (autumn), 73% (winter), 80.4% (spring), and 84.5% (summer). The abundance of Bacteroidetes in autumn and winter was higher than in others. Interestingly, <i>Epsilonbacteraeota,</i> which only exists in autumn and winter, was discovered accidentally in the midgut. We speculated that this might participate in the nutritional supply of adult mosquitoes when temperatures drop. <i>Wolbachia</i> is the most abundant in autumn, accounting for 31.6% of bacteria. The content of <i>Pantoea</i> was highest in the summer group, which might be related to the enhancement of the ability of mosquitoes as temperatures increased. <i>Pseudomonas</i> is carried out as the highest level in winter. On the contrary, in spring and summer, the genus in highest abundance is <i>Enterobacter</i>. <i>Acinetobacter</i> enriches in the spring when it turns from cold to hot. By studying the diversity of midgut bacteria of <i>Cx. quinquefasciatus</i>, we can further understand the co-evolution of mosquitoes and their symbiotic microbes. This is necessary to discuss the seasonal variation of microorganisms and ultimately provide a new perspective for the control of <i>Cx. quinquefasciatus</i> to reduce the spread of the diseases which have notably vital practical significance for the effective prevention of <i>Cx. quinquefasciatus</i>.