A recent human exome-chip study on plasma lipids identified a missense mutation in the A1CF (APOBEC1 complementation factor) gene that associates with elevated TG levels, but how A1CF influences plasma TG is unknown. Here, we evaluated whole-body A1cf knockout mice (A1cf-/-), knock-in mice homozygous for the TG-associated Gly398Ser mutation (A1cfGS/GS), and A1cf-/- rat hepatoma cell lines to elucidate A1CF's role in TG metabolism. Both A1cfGS/GS and A1cf-/- mice exhibited increased fasting plasma TG, establishing that the TG phenotype is due to A1CF loss of function. Although A1CF is known to facilitate APOBEC1’s editing of APOB mRNA to yield apoB-48, no significant shifts in apoB-100/apoB-48 were detected in the plasma, livers, and small intestines of A1cf-/- mice. To identify novel pathways for A1CF's role in TG metabolism, we performed RNA-seq of whole livers from wild-type and A1cf-/- mice and found that pro-inflammatory, not lipogenesis, genes were upregulated as a secondary effect of A1CF deficiency. Differential alternative splicing (AS) analysis revealed that genes involved in cellular stress and metabolism underwent splicing switches in A1CF deficiency, establishing a novel role for A1CF as a splicing regulator. RNA editing analysis identified in wild-type, but not in A1cf-/- mice, differential RNA editing of genes involved in protein processing and cellular stress. To evaluate the physiological impact of A1CF deficiency, we performed TG secretion and clearance studies in vivo and found that A1cf-/- mice exhibited increased TG secretion without changes in clearance. Increased VLDL-apoB secretion was also seen in A1cf-/- rat hepatoma cells, but no increase in apoB synthesis was detected on pulse-chase. Instead, proteasomal inhibition led to increased cellular stress in A1cf-/- cells, and higher expression of ER-stress protein GRP78 was detected on immunoblot of resting A1cf-/- cells, consistent with the pathways of genes affected by differential AS and RNA editing in A1CF deficiency. These data suggest an important role for A1CF in mediating VLDL-TG secretion through regulating intracellular stress.