In plants and other eukaryotes, long-chain acyl-CoAs are assumed to be imported into peroxisomes for beta-oxidation by an ATP binding cassette (ABC) transporter. However, two genes in Arabidopsis thaliana, LACS6 and LACS7, encode peroxisomal long-chain acyl-CoA synthetase (LACS) isozymes. To investigate the biochemical and biological roles of peroxisomal LACS, we identified T-DNA knockout mutants for both genes. The single-mutant lines, lacs6-1 and lacs7-1, were indistinguishable from the wild type in germination, growth, and reproductive development. By contrast, the lacs6-1 lacs7-1 double mutant was specifically defective in seed lipid mobilization and required exogenous sucrose for seedling establishment. This phenotype is similar to the A. thaliana pxa1 mutants deficient in the peroxisomal ABC transporter and other mutants deficient in beta-oxidation. Our results demonstrate that peroxisomal LACS activity and the PXA1 transporter are essential for early seedling growth. The peroxisomal LACS activity would be necessary if the PXA1 transporter delivered unesterified fatty acids into the peroxisomal matrix. Alternatively, PXA1 and LACS6/LACS7 may act in parallel pathways that are both required to ensure adequate delivery of acyl-CoA substrates for beta-oxidation and successful seedling establishment.