Activating transcription factor (ATF) 3 regulates chemokine expression in various cell types and tissues. Herein, we studied this regulation in contracting muscle cells in vitro, and in skeletal muscle after muscle-damaging exercise in vivo. C2C12 myotubes with normal or low ATF3 levels (atf3_siRNA) were electrically stimulated (EPS). Also, ATF3-knockout (ATF3-KO) and control mice ran downhill until exhaustion, and muscles were analyzed post-exercise. EPS increased ATF3 levels in myotubes (P < 0.01). Chemokine C-C motif ligand (ccl) 2 mRNA increased post-EPS, but atf3_siRNA attenuated the response (P < 0.05). Atf3_siRNA up-regulated cd6 basal mRNA, and down-regulated ccl9 and chemokine C-X-C motif ligand (cxcl)1 basal mRNAs. Post-exercise, ATF3-KO mice showed exacerbated mRNA levels of cc16 and ccl9 in soleus (P < 0.05), and similar trends were observed for cd2 and interleukin (il) 1 beta (P < 0.09). In quadriceps, il6 mRNA level increased only in ATF3-KO (P < 0.05), and cxcl1 mRNA showed a similar trend (P = 0.082). Cluster of differentiation-68 (cd68) mRNA, a macrophage marker, increased in quadriceps and soleus independently of genotype (P < 0.001). Our data demonstrate that ATF3 regulates chemokine expression in muscle cells in vitro and skeletal muscle in vivo, but the regulation differs in each model. Cells other than myofibers may thus participate in the response observed in skeletal muscle. Our results also indicate that ATF3-independent mechanisms would regulate macrophage infiltration upon muscle damaging exercise. The implications of chemokine regulation in skeletal muscle remain to be determined. (C) 2017 Elsevier Inc. All rights reserved.