β-Carotene has been reported to alleviate hepatic steatosis (SS), inflammation, and fibrosis in vivo and vitro studies. However, the clinical relevance of serum β-carotene and β-carotene-to-retinol (SC/SR) ratio with histological severity in nonalcoholic fatty liver disease (NAFLD) patients is unknown. This case-control study enrolled 24 control subjects and 62 NAFLD patients. Liver biopsies were collected and histological characteristics were assessed. Information with regard to demographic, anthropometric and dietary intake was assessed by face-to-face interviews with questionnaire. Serum β-carotene and retinol concentrations were determined by the HPLC method. Serum β-carotene and SC/SR levels in NAFLD patients were significantly lower than these in controls (0.23 ± 0.01 vs. 0.35 ± 0.04 μmol/L, 0.38 ± 0.03 vs. 0.84 ± 0.10). Compared with individuals without SS, both β-carotene and SC/SR levels were significantly decreased in those with moderate SS (0.34 ± 0.03 vs. 0.21 ± 0.02 μmol/L, 0.76 ± 0.09 vs. 0.37 ± 0.05). Subjects with mild inflammation had a significantly lower β-carotene and SC/SR levels than those without inflammation (0.23 ± 0.01 vs. 0.33 ± 0.04 μmol/L, 0.77 ± 0.09 vs. 0.38 ± 0.03). Serum SC/SR was significantly lower in patients with mild fibrosis than those without fibrosis (0.45 [0.27–0.83] vs. 0.34 [0.26–0.51]). According to the NAFLD Activity Scoring score, both β-carotene and SC/SR gradually decreased with disease progression from normal liver, simple SS to steatohepatitis borderline (ptrend ≤ 0.001). These results show that NAFLD patients have lower circulating β-carotene concentration and SC/SR ratio, which are associated with the histological severity of NAFLD.