In this study, we conducted a 56-d feeding trial to investigate the effects of replacing the fish oil (FO) with palm oil (PO) on the performance, tissue fatty acid (FA) composition, and mRNA levels of genes related to hepatic lipid metabolism in grouper (
Epinephelus coioides
). Five isolipidic (13% crude lipid) and isonitrogenous (48% CP) diets were formulated by incrementally adding PO to the control diet (25% fish meal and 9% added FO) to replace FO in the control diets. Triplicate groups of 30 groupers (initial weight: 12.6 ± 0.1 g) were fed one of the diets twice daily, to apparent satiety. The replacement of FO with 50% PO revealed maximum growth without affecting the performance and whole-body proximate compositions, and replacing FO with 100% PO revealed a comparable (
P
> 0.05) growth with that of the control diet, suggesting PO as a suitable alternative to FO. The analysis of FA profiles in the dorsal muscle and liver though reflected the FA profile of the diet, PO substitutions above 50% could compromise (
P
< 0.05) the FA profile in the liver and flesh of the fish species in comparison with the control diet. Furthermore, the mRNA levels of
FAS
,
G6PD
,
LPL
,
PPARΑ
, and
Δ6FAD
genes in the liver had positive linear and/or quadratic responses, but the
SCD
,
HSL
,
ATGL
,
FABP
,
SREBP-1C
and
ELOVL5
had the opposite trend, with increasing dietary PO inclusion levels, whereas the mRNA level of
ACC
was not affected by dietary treatments. The optimal level of PO substitution for FO was estimated to be 47.1% of the feed, based on the regression analysis of percent weight gains against dietary PO inclusion levels; however, it might affect the FA profile in the liver and flesh of the fish species, and further study is required to investigate whether the changes in tissue FA composition will affect the welfare and market value over a production cycle of grouper.