Spotting disease is common in cultured sea urchin (Strongylocentrotus intermedius) from China, and outbreaks are most frequent in summer when seawater temperatures exceed 20 °C. In this study, we used 16S rRNA and housekeeping gene sequence alignments to characterize two strains of Vibrio sp. from diseased sea urchins in a North China farm in September 2017. The strains included a new strain of Vibrio owensii (named HD-1) and a new Vibrio species (named HD-2). Phylogenetic analysis indicated that HD-1 and HD-2 are located in different evolutionary clades and exhibited a high degree of divergence. Transmission electron microscopy showed morphological differences between HD-1 and HD-2. Laboratory-based challenge tests showed that sea urchins infected with either HD-1 or HD-2 exhibited pathological symptoms of spotting disease in a dosage- and temperature-dependent manner. The virulence of HD-1 was greater than that of HD-2. Antimicrobial susceptibility tests demonstrated that both HD-1 and HD-2 are sensitive to ciprofloxacin and florfenicol. HD-1 is specifically sensitive to neomycin, and HD-2 is specifically sensitive to cephalexin.