Abstract: Greenhouse cultivation is the most common method of vegetable production worldwide, and cucumber is one of the most important greenhouse vegetables. However, continuous cucumber cropping in intensive greenhouse production causes a high incidence of Fusarium wilt. It has been demonstrated that the occurrence of this disease was correlated with the alteration in plant rhizosphere microbiome. However, previous studies have focused on the overall microbial community (i.e., bacteria and fungi). The potential role of functional rhizosphere microorganisms in disease occurrence remains largely unclear. Nitrite oxidation (NO₂⁻ conversion to NO₃⁻), performed by nitrite-oxidizing bacteria (NOB), is a vital process in soil nitrification and therefore affects soil N availability and plant nitrogen uptake. In this study, we targeted greenhouse cucumbers subjected to continuous cropping by using rhizosphere soil samples from healthy plants (HPR) and Fusarium wilt-diseased plants (DPR), and assessed differences in their abundances and community diversities and structures of two major groups of NOB, Nitrobacter and Nitrospira, using real-time quantitative PCR and high-throughput amplicon sequencing. The results showed that there was no significant difference in the NO₂⁻-N content between DPR and HPR, whereas the potential nitrite oxidation rate (PNOR) in DPR was approximately twice as high as that of HPR (P<0.05). Nitrobacter abundance in the DPR was significantly higher than in the HPR (P<0.05), with no significant difference in Nitrospira abundance. Nitrobacter abundance was significantly and positively correlated with PNOR, suggesting that it dominated soil nitrite oxidation. For both Nitrobacter and Nitrospira, community diversity did not differ between the DPR and HPR, whereas significant differences in community structures were observed (P<0.05). Phylogenetic analyses revealed that the main members of the Nitrobacter community were Nitrobacter Cluster 3, Cluster 3-like, Cluster 2b, Cluster 4, Cluster 6, Cluster 1, and Cluster 5; in the Nitrospira community, Namibia soil Cluster 1, Cluster 2, Cluster 3, Nitrospira lineageⅠ, lineageⅡ, and lineageⅤ, respectively. In the Nitrobacter community, the average relative abundance of Nitrobacter Cluster 2b in HPR was significantly higher than that in DPR; and on the contrary for both Nitrobacter Cluster 6 and Nitrobacter Cluster 5. In the Nitrospira community, the average relative abundance of Namibia soil cluster 1 in the DPR significantly outnumbered that of the HPR by 92.19%, but the average relative abundance of Nitrospira lineageⅡ was far lower than that of the HPR. Redundancy analysis indicated that the NO₂⁻-N content was the most important soil physicochemical variable influencing the community structures of both Nitrobacter and Nitrospira. Among all the community members detected in this study, in terms of their average relative abundance, only Nitrobacter Cluster 6 was significantly positively linked with PNOR, suggesting that it may be an active member performing nitrite oxidation in continuously cropped greenhouse soil. Collectively, the present study confirmed that the occurrence of Fusarium wilt disease in greenhouse cucumbers in a continuous cropping system was accompanied by shifts in the community structure of NOB in the plant rhizosphere, which obviously affected nitrogen turnover in the diseased greenhouse soil.