Palmitoleic acid shows a variety of beneficial properties to human health. In this study, enrichment of palmitoleic acid from sea buckthorn pulp oil by crystallization and molecular distillation was investigated. Sea buckthorn pulp oil was first converted to its corresponding mixed fatty acids (SPOMFs) that contained 27.17% palmitoleic acid. Subsequently, the effect of various factors on crystallization (i.e., crystallization temperature, solvent, ratio of SPOMFs to solvent (w/v), crystallization time) and molecular distillation (distillation temperature) were assessed on a 5-g scale. It was found that optimal crystallization conditions were a 1:15 ratio of SPOMFs to methanol (w/v) at -20 °C for 12 h, while the optimal temperature for molecular distillation was 100 °C. These conditions were utilized to obtain a liquid oil comprising 54.18% palmitoleic acid with an overall yield of 56.31%. This method has great potential for adoption by the food and medical industries for the preparation of palmitoleic acid concentrate for nutritional studies.