With the shortage of common vegetable fat sources, such as soybean oil (
SBO
), it is urgent to find alternative oil sources for broiler producers. The objective of this study was to evaluate the potential of refined cottonseed oil (
CSO
) as a replacement for
SBO
in broiler diets. A total of 180 chickens at 1 d of age were randomly assigned to five treatments of six replicates. One treatment was the basal diet (control), and the other four experimental diets were formulated from the basal diet by replacing (w/w) 25%, 50%, 75%, and 100% of the
SBO
with refined
CSO
(only containing 0.2% cyclopropenoid fatty acids, and no free gossypol was detected). At the end of week 6, blood samples were obtained from the jugular vein and the breast muscle was aseptically isolated from two birds per replicate. The results showed that substitution of
CSO
for low‐level
SBO
had no significant effect (
p
>
0.05) on broiler performance during the starter period (week 1–3), while 50% level of
CSO
inclusion significantly increased (
p
<
0.05)
ADG
and improved
FCR
compared with the control group during the finisher period (week 4–6). Broilers fed 100%
CSO
diets had lower (
p
<
0.05) levels of serum total protein (
TP
), albumin (
ALB
), cholesterol (
CHO
) concentrations, and serum alkaline phosphatase (
AKP
) activity than that of the control broilers. Furthermore, the serum antioxidant status appeared to be enhanced by
CSO
. Additionally, high levels of
CSO
(75 and 100%) significantly increased the proportions of C14:0 and C18:0 but decreased the proportions of C18:1n9t, C18:2n6c, and ∑ n‐6 polyunsaturated fatty acids in breast muscles of broilers. Overall, the
SBO
could be replaced with refined
CSO
up to 50% in diets for broilers without adversely affecting the performance, liver functions, and breast muscle fatty acid composition of these broilers.