Purpose This study aimed to evaluate the mid-term efficacy and safety of Escherichia coli -derived bone morphogenetic protein-2 (E.BMP-2)/hydroxyapatite (HA) in lumbar posterolateral fusion (PLF). Methods This multicenter, evaluator-blinded, observational study utilized prospectively collected clinical data. We enrolled 74 patients who underwent lumbar PLF and had previously participated in the BA06-CP01 clinical study, which compared the short-term outcomes of E.BMP-2 with an auto-iliac bone graft (AIBG). Radiographs and CT scans were analyzed to evaluate fusion grade at 12, 24, and 36 months. Visual analog scale (VAS), Oswestry disability index (ODI), and Short Form-36 (SF-36) scores were measured preoperatively and at 36 months after surgery. All adverse events in this study were assessed for its relationship with E.BMP-2. Results The fusion grade of the E.BMP-2 group (4.91 ± 0.41) was superior to that of the AIBG group (4.25 ± 1.26) in CT scans at 36 months after surgery ( p  = 0.007). Non-union cases were 4.3% in the E.BMP-2 and 16.7% in the AIBG. Both groups showed improvement in pain VAS, ODI, and SF-36 scores when compared to the baseline values, and there were no statistically significant differences between the two groups. No treatment-related serious adverse reactions were observed in either group. No neoplasm-related adverse events occurred in the E.BMP-2 group. Conclusions The fusion quality of E.BMP-2/HA was superior to that of AIBG. E.BMP-2/HA showed comparable mid-term outcomes to that of AIBG in terms of efficacy and safety in one-level lumbar PLF surgery.