Recently, a new bacterial disease was detected on cucumber stalks. In order to study the pathogenesis of this disease, the pathogenic bacteria were isolated and identified on the basis of morphological and molecular characteristics, and further analyzed for pathogenicity and antagonistic evaluation. Pathogenicity analysis showed that HlJ-3 caused melting decay and cracking in cucumber stems, and the strain reisolated from re-infected cucumber stalks was morphologically identical to HlJ-3 colonies, which is consistent with the Koch's postulates. The pathogenic strain HlJ-3 was identified as having similar morphological characteristics to Bacillus subtilis. Meanwhile, its internal transcribed spacer sequence (ITS) and DNA gyrase A subunit (gyrA) were both more than 99% homologous and clustered on the same branch with B. subtilis. Therefore, combined with morphological and molecular biological features, strain HlJ-3 was identified as B. subtilis. In addition, B. subtilis, which has a wide range of hosts, was able to infest other common crop species, including potato, tomato, pepper, melon, and radish. Furthermore, antagonistic evaluation confirmed that strain HlJ-3 strongly inhibited the mycelial growth of Colletotrichum coccodes and Alternaria tenuissima in vitro, with antagonistic effects of 69.92% and 68.08%, respectively. In conclusion, our results showed that strain HlJ-3 is B. subtilis, which is pathogenic to cucumber in vivo and can infect plants of Solanaceae, Cucurbitaceae and Brassicaceae with a wide range of hosts. In addition, this strain has good biocontrol effects against C. coccodes and A. tenuissima in vitro. The findings of this research will help to prevent and control the occurrence of this pathogen and regulate its use as a biocontrol agent.