Abstract Spiders frequently suffer abnormal molting and subsequent death when sustained on a monotypic diet of fruit flies Drosophila melanogaster. The fruit flies lack arachidonic acid (AA), a nutrient that may be critical for successful molting of spiders. To test this hypothesis, we compared the survival rates of juvenile wolf spiders, Pardosa pseudoannulata fed on three kinds of diets, midges (Tendipes sp.), fruit flies enriched in AA and fruit flies lacking in AA. In addition, we analyzed the fatty acid composition of spiders feeding on midges and fruit flies enriched in AA and their prey. Juvenile spiders fed on midges and AA-enriched fruit flies reached full maturity without molting issues, whereas nearly all spiders fed the flies lacking in AA did not survive. Among the 35 different fatty acids, only the AA levels in both prey and spiders correlated positively with spider survival. Our findings provide compelling evidence that AA is crucial for the survival and molting of P. pseudoannulata.