Background: Although neoadjuvant chemotherapy (NACT) has been widely investigated, the magnitude of the clinical benefit and the potential risk of NACT followed by surgery compared with upfront surgery remains unclear for patients with locally advanced pancreatic cancer (LAPC). Methods: This retrospective, prospective cohort-based analysis included 135 patients who underwent NACT followed by surgery and 359 patients who received upfront surgery for LAPC between October 2005 and April 2017. Disease-free survival (DFS) and overall survival (OS) from surgery were compared between the two groups. Results: There were no significant differences in gender (male, 53% vs 56%) and age (median 60 vs 61 years) between the NACT followed by surgery group and upfront surgery group. As NACT, gemcitabine-based regimens and FOLFIRINOX were used in 69 (51%) and 66 (49%) patients, respectively. The NACT followed by surgery group showed significantly less advanced T stage (T3–4, 93% vs 99%, p = 0.001) and N stage (N+, 49% vs 71%, p < 0.001) than the upfront surgery group. NACT followed by surgery was significantly associated with better OS (median, 25.4 [18.6–32.2] vs 17.1 [15.5–18.7] months, p = 0.001) and DFS (median, 9.0 [95% CI, 6.8–11.2] vs 7.1 [6.4–7.8] months, p = 0.005) than upfront surgery. These results were consistent in the multivariate analysis for OS (adjusted hazard ratio [aHR], 0.73 [95% CI, 0.56–0.96], p = 0.02) and DFS (aHR, 0.72 [95% CI, 0.56–0.93], p = 0.01). There was no difference in length of hospital stay (median 13 vs 17 days, p = 0.14) for surgery between the two groups, and the NACT followed by surgery group showed a significantly lower incidence of postoperative complication than the upfront surgery group (38% vs 27%, p = 0.03). Conclusions: The present study revealed that NACT followed by surgery may provide survival benefit compared with upfront surgery in LAPC without causing significant safety issues.