Background: Pathological ocular neovascularization, a characteristic feature of proliferative ocular diseases, is a primary contributor to global vision impairment. The dynamics of tubulin are crucial in maintaining ocular homeostasis, closely linked to cellular proliferation and angiogenesis. Elucidating the molecular mechanisms driving this process is vital for formulating effective therapeutic strategies. Methods: Multiple transcriptome analyses revealed upregulation of endothelial tubulin beta-6 chain (Tubb6) in oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) mice models. Transwell migration assay, wound healing assay, tube formation assay, flow cytometry, and immunofluorescent staining were employed to identify the role of TUBB6 knockout (KO) in vitro. The effects of Tubb6 silencing on retinal angiogenesis and choroidal neovascularization were subsequently evaluated. Results: We identified upregulated Tubb6 expression in retinas from OIR mice through combination analyses of single-cell RNA sequencing (scRNA-Seq) and bulk RNA-Seq. The RNA expression profiles of endothelial cells (ECs) from proliferative diabetic retinopathy (PDR) patients and neovascular age-related macular degeneration (nAMD) patients also exhibited an elevation in TUBB6. Notably, Tubb6 was abundantly expressed in ECs and pericytes, and was predominantly localized to proliferative ECs and vascular tip cells. Functional studies demonstrated that TUBB6 knockdown reduced the expression of proliferative and tip cell markers in vitro. Tubb6 deficiency decreased vascular sprouting and tip cell formation of OIR mice retina and retarded CNV progression in vivo. Mechanistically, YBX1, an RNA-binding protein, was identified as an upstream regulator of TUBB6 via binding to its 3' untranslated region (3'UTR) and maintaining mRNA stability. Transcriptome analysis further linked TUBB6 to the activity of WNT pathway. TUBB6 silencing suppressed the WNT signaling pathway, with WNT3A and FZD8 identified as downstream targets. Conclusions: Collectively, our research shed light on the pivotal function of TUBB6 in maintaining ocular homeostasis and uncovered the YBX1-TUBB6-WNT3A/FZD8 pathway's involvement in sprouting angiogenesis. Targeting TUBB6 and developing its specific inhibitor could pioneer new approaches for treating ocular microvascular diseases.