Altered gut microbial ecology contributes to the development of metabolic diseases including obesity. However, studies based on different populations have generated conflicting results due to diet, environment, methodologies, etc. The aim of our study was to explore the association between gut microbiota and BMI in Chinese college students. The 16S next-generation sequencing (NGS) was used to test the gut microbiota of nine lean, nine overweight/obesity and ten normal-weight male college students. The differences in gut microbiota distribution among three groups were compared, and the relationship between the richness, diversity, composition of gut microbiota and BMI were analysed. The predominant phyla Bacteroidetes and Firmicutes were further confirmed by real-time PCR. Metagenomic biomarker discovery was conducted by linear discriminant analysis (LDA) effect size (LEfSe). NGS revealed that gut microbiota composition was different among three groups, but there was no difference in the abundance ratio of Firmicutes:Bacteroidetes. Several bacterial taxa were in linear relationship with BMI (positive relationship: uncultured bacterium (Bacteroides genus); negative relationship: Porphyromonadaceae, Acidaminococcaceae, Rikenellaceae, Desulfovibrionaceae, Blautia, Anaerotruncus, Parabacteroides, Alistipes). Moreover, gut microbiota diversity decreased with the increase in BMI. And LEfSe analysis indicated that Blautia, Anaerotruncus and its uncultured species were significantly enriched in the lean group (LDA score ≥ 3), Parasuterella and its uncultured species were significantly enriched in the overweight/obese groups (LDA score ≥ 3). In general, gut microbiota composition and microbial diversity were associated with BMI in Chinese male college students. Our results might enrich the understanding between gut microbiota and obesity.