Cis (Z)-β-carotenes with 25.3% 9Z-β-carotene were prepared for nanostructured lipid carriers (NLCs). The optimal conditions for NLC preparation using an orthogonal experimental method were as follows: the total lipid concentration was 9% (w/v), the surfactant concentration was 1.4% (w/v), the solid to liquid lipid ratio was 3:1 (w/w), and the homogenization pressure was set at 500 bar for three cycles. Under these conditions, the encapsulation efficiency (%) of the NLC was 95.64%, and the total β-carotene in NLCs was 2.9 mg/mL, which was significantly higher than those reported by others. The proportion of total Z-β-carotenes was as high as 53.3%, the particle size was 191 ± 6.46 nm, and the polydispersity index was 0.2 ± 0.03. Storage stability results indicated that the β-carotene-loaded NLC stabilizes both 9Z-β-carotene and total β-carotene from leakage and degradation during 21 days of storage at pH 3.5–7.5 at low temperatures (4 °C), especially for the more bioactive 9Z-β-carotene. The technique with an improved ratio of 9Z-β-carotene, loading capacity, water solubility, and bioaccessibility of the β-carotene NLC provides an effective strategy for β-carotene applications in functional foods or beverages and in nutraceutical preparations.