Streptococcus suis is an important zoonotic pathogen that can cause severe infections in both humans and pigs. The prevalence of S. suis serotype 14 in sporadic cases in China has been gradually increasing during recent years. However, the current understanding of S. suis serotype 14 is limited. In this study, we investigated the population structure, phylogenetic relationships, antimicrobial resistance, and pathogenicity of 125 S. suis serotype 14 strains. These strains clustered into 12 sequence types (STs) and three clonal complexes (CCs), with ST7 accounting for the highest proportion (73.6%), which indicates significant pathogenic potential, given that ST1 and ST7 are well-known high-virulence STs in S. suis. Bioinformatic analysis showed that all serotype 14 strains carry the virulence genes sly and epf, while 74.4% of the strains carry the virulence gene mrp. In the pathogenicity test (n = 5), the human strain Ss2301, Ss2401, and the porcine strain L966, SC42 proved to be highly virulent strains. These data highlight the virulence potential of serotype 14 S. suis. Tetracycline resistance genes and macrolide, lincosamide, and streptogramin B (MLSB) resistance genes were most frequently detected in the population. The transmission of the former genes mainly depends on integrative and conjugative elements (ICEs), while the latter depends on both ICEs and prophages. This study not only confirmed the pathogenic potential of serotype 14 S. suis but also provided valuable information for improving prevention and control strategies for S. suis infections.